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Nitrogenase XII. Mössbauer studies of the MoFe protein from Clostridium pasteurianum W5
124
Biochimica et Biophysica Acta, 623 (1980) 124--138
© Elsevier/North-Holland Biomedical Press
BBA 3 8 3 9 4 NITROGENASE XII MOSSBAUER STUDIES OF THE MoFe PROTEIN FROM C L O S T R I D I U M P A S T E U R I A N U M W5 B.H. HUYNH a, M.T. HENZL b, J.A. CHRISTNER a, R. ZIMMERMANN c, W.H. ORME-JOHNSON b and E. MUNCK a a Gray Freshwater Biological Institute/Department of Biochemistry, University of Minnesota, Navarre, MN 55392, b Department of Biochemistry, Center for Studies of Nitrogen Fixation, College of Agricultural and Life Sciences, University of Wisconsin, Madison, WI 53706 (U.S.A.) and c Physikalisches Institut, University of Erlangen, D-8520 Erlangen (F.R.G.)
(Received September llth, 1979) Key words: Nitrogenase; MSssbauer spectroscopy; MoFe protein; (C. pasteurianum)
Summary We have studied the molybdenum-protein (MoFe protein) from Clostriwith MSssbauer spectroscopy in the temperature range from 1.5 to 200 K in magnetic fields up to 55 kG. Except for some small differences in the hyperfine parameters the results for the C. p a s t e u r i a n u m prorein are essentially the same as those published previously for the protein from A z o t o b a c t e r vinelandii, i.e. (30 + 2) Fe atoms partition into t w o identical cofactor centers M (each center most likely containing six Fe atoms and one Mo atom), four P-clusters (each center containing four Fe atoms), and one iron environment labeled S (about t w o Fe atoms per holoenzyme). We have analyzed the spectra of the cofactor centers in three distinct oxidation states, dium pasteurianum
e--
e-
M O X ~ MN - - , MR . The diamagnetic (electronic spin S = 0) state M ° x is attained by oxidation of the native, EPR-active (S = 3/2) state M N. The reduce~t state M R is observed in steady state under nitrogen fixing conditions; high-field MSssbauer studies show that the cofactor centers are paramagnetic (integer electronic spin S i> 1) in the state M R. We have evaluated the complex high-field spectra resulting from the P-clusters in the oxidized state pOX. The analysis shows that one iron site is characterized by a positive hyperfine coupling constant A0 while the other three sites have A0 < 0. A slightly modified set of parameters also fits the high-field data of the MoFe protein from A. vinelandii. Finally, we will present a discussion summarizing our principle results obtained to date for the proteins from A. vinelandii and C. p a s t e u r i a n u m .
125 Introduction
A current and widely held working hypothesis [1--4] about the mechanism of nitrogenase is that MgATP hydrolysis-driven electron transfer from the Fe protein leads to reduced redox centers in the MoFe protein. These are subsequently reoxidized during the reduction of nitrogenase by substrates such as H ÷, C2H2 and N2. In parts VIII [5], X [6], and XI [7] of this series we have examined the MoFe protein from Azotobacter vinelandii, using combined EPt~ and MSssbauer spectroscopy, in an effort to characterize the redox centers in the protein. We believe that in all essential respects MoFe protein from any source will be found to have the same catalytic groups; we have previously commented [ 5,6] on the similarity of MSssbauer spectra from Klebsiella pneumoniae and A. vfnelandii MoFe proteins. As part of a limited survey of the physical properties of nitrogenases, and because the Clostridium pasteurianum enzyme has played a central role in the development of the biophysical picture of nitrogenase [ 1,8], we undertook to study this protein in some detail. Materials and Methods C. pasteurianum W5 was grown on a nitrogen-free medium in 18 1 cultures. The medium contained (per 1): 20 g sucrose, 0.1 M potassium phosphate (pH 7.6), 20 mM potassium citrate, 10 mg NaC1, 50mg CaC12 • 2 H20, 0.5 g MgSO4 • 7 H20, 20 mg Na2MoO4 • 2 H20, 1 mg STFe, and 50 Izg biotin. The potassium phosphate was added as a 1 M solution prepared from reagent grade KOH and H3PO4. Contaminating S6Fe was removed from the sucrose and phosphate solu. tions by autoclaving them in the presence of A1203 [9] and filtering after they had been allowed to stand for several days at 4°C. The doubling time for the organism at 30°C on this medium was 120 min. Nitrogen was bubbled through the culture at 2--3 1/min. When the absorbance of the culture (measured at 650 nm in a 1 cm pathlength cuvette on an aliquot of the culture, appropriately diluted such that the spectrophotometer reading was less than 0.4) reached 1.8, the culture was harvested with the aid of a small Sharples centrifuge. The cell paste (50--75 g) was immediately frozen in liquid nitrogen. Nitrogenase was purified as follows: Aerobic buffer (25 mM Tris-HC1, pH 8.5, at 30°C) was added to frozen C. pasteurianum cell paste in a weight ratio 3 : 1 . Lysosyme (1 mg/cell paste) and deoxyribonuclease I (0.1 mg/g cell paste), both from Worthington, were added. The resulting suspension was shaken under hydrogen for 1 h at 30°C. Cell debris was removed by centrifuga. tion (11 000 × g) for 45 rain. After equilibrium the supernatant with CO (0.2 atom in a sealed suction flask) and adding dithionide, it was loaded onto an anaerobic column of DEAE-cellulose (Whatman DE32). After washing the column with 0.15 NaC1 the MoFe protein and the Fe protein were eluted with 0.25 and 0.4 M NaC1, respectively. Both solutions were then equilibrated with CO, which serves to inhibit chlostrial hydrogenase. All chromatographic steps ~n the isolation were preformed at 15 ° C. The clostridium MoFe protein fractions was further purified by chromatography on Sephadex 6B (Pharmacia), equilibrated with 50 mM Tris-HC1, pH 8.0 (30°C). The fractions of highest specific activity were then concentrated and
126
subjected to chromatography on DEAE-cellulose, employing a NaC1 gradient (0.15 M--0.30 M) for elution. The most active fractions were combined and concentrated. The Fe protein fraction was further purified by chromatography on Sephadex G-100 (Pharmacia), equilibrated with 50 mM Tris-HC1, pH 8.0, containing 20% (v/v) glycerol. The STFe-enriched MoFe protein preparation used in these experiments had a protein concentration of 37 mg/ml, reduced 1200 nmol C2H2 per min per mg, and contained 22 gatoms of Fe per mol. The Fe protein solution contained 14 mg protein per ml and had a specific activity of 2200 nmol • min-' • mg-'.
Nitrogenase during turnover -- preparation o f MiSssbauer samples (a) Manual mixing. 0.58 ml clostridium MoFe protein (97 nmol) was combined with 0.42 ml clostridium Fe protein (103 nmol) in a N2-filled serum bottle. 0.025 ml of 1 M Na2S204 in 0.1 M Tris base was added, and the solution was incubated for 5 min at 0°C. Then 0.2 ml of cold (0°C) anaerobic MgATP generator was introduced by syringe. 30 s after adding the generator, the reaction mixture was injected into a MSssbauer cuvette, housed in a N2-filled loading chamber which had been pre-cooled with liquid nitrogen. The sample was frozen within 2 min after initiating the reaction. In a control experiment monitored with EPR spectroscopy, we demonstrated that, at 0°C, the height of the g = 3.78 feature of the MoFe protein spectrum had decreased by 87% after 30 s of reaction. The MgATP generating system was prepared by dissolving 120 mg disodium A T P - 3 H20 (P-L Biochemicals) and 530 mg creatine phosphate (Pierce Chemical Co.) in 1 ml of glass-distilled water, and the pH was adjusted to 7. Then 15 mg of creatine phosphokinase (Sigma) and 300 pl of 1 M Mg(CH3COO)2 • 4 H20 were added, and the volume was adjusted to 2.0 ml. (b) Rapid mixing. 0.30 ml of clostridium MoFe protein was rapidly mixed with 0.15 ml of clostridium Fe protein to give final concentrations of 120 and 80 #M, respectively, for the two proteins. The syringe containing the clostridium Fe protein solution also contained Mg2÷, disodium ATP • 3 H20, creatine phosphate, and creatine phosphokinase at levels such that, after mixing the following concentrations were achieved: 2.5 mM Mg2÷, 2.5 mM ATP, 75 mM creatine phosphate, 13 mg/ml creatine phosphokinase (1900 I.U./ml). After incubation at 30°C for 8 s, the reaction mixture was shot into cold (120 K) isopentane. After the frozen material had settled, it was packed into a MSssbauer cuvette. A parallel sample collected for examination by EPR spectroscopy revealed that the height of the g = 3.78 signal had decreased by at least 90%. The Mbssbauer spectrometer at the Gray Freshwater Biological Institute and the methodology of Mbssbauer data analyses have been described previously [6,10]. Results
For background information about the P-clusters and the M-centers (cofactor centers) the reader is referred to the literature [5--7,11]. Fig. 1 summarizes the various oxidation states which have been attained and which we have characterized for azotobacter MoFe protein. The nomenclature of the MSssbauer components is given in Table I of Ref. 6.
127 i
O.0 4.2K
I
.........' ",,'
I--- 3 . 0 Z bJ U tY W
COFACTOR
CENTER M
M"
M ox
MR
I
z__o.o .................................... z_o
I
,:............... ,' ,%
, , ,,,' , , /
Fe"
_ ..........2
i---
e-
e-..
S=3~
S=O
~ m 3£ S~1
(EPR active) F-
,
""
P- CLUSTER 27K
pOX
"%",
.....",,, ,,,,,:
,,,'
p.
e-.
~, --~--->,Fe '÷
I
/ .D--/-;S
S_>~ (EPR silent )
S=O
I
-2
i
0 2 VELOCITY ( m m / s )
,
I
4
Fig. 1. S t a b l e o x i d a t i o n s t a t e s o f t h e c o f a c t o r c e n t e r s a n d t h e P - c l u s t e r s . T h e c u b a n e 4 F e - 4 S s t r u c t u r e d e p i c t e d f o r t h e P - c l u s t e r is v e r y p r o b a b l e , b u t n o t a n e s t a b l i s h e d f a c t . T h e l e t t e r s D s y m b o l i z e t h r e e i r o n a t o m s w h i c h give rise t o t h e q u a d r u p o l e d o u b l e t l a b e l e d D ; t h e F e 2+ site y i e l d s t h e d o u b l e t l a b e l e d F e 2+. Fig. 2. M 6 s s b a u e r s p e c t r a o f n a t i v e M o F e p r o t e i n f r o m C. pasteurianurn W 5 t a k e n in a p a r a l l e l field o f 6 0 0 G. T h e s p e c t r a i n ( A ) a n d (B) w e r e o b t a i n e d a t 4 . 2 K a n d 7 0 K , r e s p e c t i v e l y . T h e s o l i d Hne i n (B) is the result of fitting four doublets to the data with the constraints described in the text. The deconvoluted s p e c t r u m i n (C) w a s o b t a i n e d as d i s c u s s e d f o r t h e a z o t o b a c t e r M o F e p r o t e i n [ 7 ] .
Results for the native MoFe protein Fig. 2A shows a spectrum of STFe enriched clostridium MoFe protein taken at 4.2 K in a parallel field of 600 G. (We have recorded approx. 150 spectra of t w o clostridium MoFe protein batches in the temperature range from 1.5 to 240 K in fields up to 55 kG). Fig. 2B shows a spectrum of the same sample taken at 70 K. In Fig. 2 the t w o quadrupole doublets D and Fe 2÷ (the signature of the P-clusters) are clearly discerned. C o m p o n e n t M N, the spectrum resulting from the cofactor centers in their EPR active state, appears at 4.2 K as a magnetic spectrum (shown in Fig. 3 below). A t 70 K the electronic spin S = 3/2 of the cofactor centers relaxes rapidly; the magnetic hyperfine interactions are averaged o u t and c o m p o n e n t M N appears as a quadrupole d o u b l e t in Fig. 2B. To re¢olve c o m p o n e n t S optimally we have used the same procedure as described for azotobacter MoFe protein [7]: the spectrum was recorded at a temperature (27 K) where c o m p o n e n t M N is strongly broadened by relaxation, and then deconvoluted. The deconvoluted spectrum demonstrates the presence
128
of S and it defines the line positions and widths of the doublets, D, Fe 2÷, and S quite precisely. The shape of c o m p o n e n t M N at 70 K was obtained by subtracting the spectrum of Fig. 2B from that of Fig. 2A. Since the MSssbauer parameters of D, Fe 2÷ and S are independent of temperature for T < 100 K the contributions of these species cancel. The resulting difference spectrum is the magnetic spectrum M s superimposed with an inverted doublet, the M s spectrum at 70 K. The lineshape of M E is somewhat non-Lorentzian (12 Fe/molecule contribute!) and has a full width at half max. of a b o u t 0.33 mm/s. For the (average) quadrupole splitting and isomeric shift we found AEQ = 0.81 mm/s a n d ~ = 0.41 mm/s (with respect to metallic Fe), respectively. With a precise knowledge of all line positions and good information a b o u t their widths we have least-squares fitted the spectrum in Fig. 2B assuming four quadrupole doublets. This procedure yields a reliable value for the fraction at the Fe 2÷ sites (12.8%), in agreement with the results on azotobacter MoFe protein (13%). The relative intensities of the D and M s doublets could n o t be reliably determined with this procedure primarily because M s has somewhat non-Lorentzian lines. We have therefore fixed the intensity of doublet D by assuming that the D and Fe 2÷ sites occur in the ratio of 3 : 1. This yields an intensity for D consistent with (a) the results on azotobacter MoFe protein, (b) the requirements for preparing the spectra of Fig. 3 (the 4.2 K spectra show that the absorption of D
0.0 0.~
'
I
'
A ~'~
I
0 . 6 kG
i
\
I
,
'
~
I..,nr
m
<
-4
-2
U L V E L O C I T Y (m rn/s )
"*
Fig. 3. L o w - t e m p e r a t u r e M ~ s s b a u e r s p e c t r a o f the c o f a c t o r c e n t e r s M i n 6 0 0 G p a r a l l e l (A), 6 0 0 G t r a n s verse (B), a n d 5 0 k G p a r a l l e l (C) m a g n e t i c fields. T h e s p e c t r a w e r e o b t a i n e d f r o m t h e r a w d a t a b y r e m o v i n g the c o n t r i b u t i o n s o f D , F e 2+, a n d S. B e c a u s e o f the large c o n t r i b u t i o n o f c o m p o n e n t D the shapes o f t h e 'experimental' spectra in ( A ) a n d (B) a r e n o t v e r y well d e f i n e d in t h e v e l o c i t y r a n g e f r o m - - 0 . 5 m m / s t o + 1 . 5 m m / s . T h e s o l i d lines a r e t h e o r e t i c a l spectra generated w i t h the parameter set listed in T a b l e II. A l m o s t i d e n t i c a l t h e o r e t i c a l s p e c t r a o f the s u b c o m p o n e n t s A 1 , A 2 , A 3 , a n d B ( t h r e e i d e n t i c a l s i t e s ) h a v e b e e n p l o t t e d previously for a z o t o b a c t e r M o F e p r o t e i n [ 7 ] . T h e a g r e e m e n t b e t w e e n e x p e r i m e n t a n d t h e o r y i n C c o u l d be i m p r o v e d b y a l l o w i n g d i f f e r e n t h y p e r f i n e p a r a m e t e r s for the three B - c o m p o n e n t s .
129 TABLE I M{'~SSBAUER P A R A M E T E R S PROTEIN
FOR
CLOSTRIDIUM
MoFe PROTEIN
AND AZOTOBACTER
MoFe
The data on azotobacter MoFe protein are taken from Ref. 5 and 7. Isomeric shifts are quoted at 4.2 K relative t o m e t a l l i c i r o n a t r o o m t e m p e r a t u r e . T h e n u m b e r s in p a r e n t h e s e s give t h e e r r o r in t h e l a s t signific a n t digit. If n o u n c e r t a i n t i e s a r e q u o t e d , t h e p a r a m e t e r s a r e a v e r a g e v a l u e s o f u n r e s o l v e d s u b e o m p o n e n t s . For both azotobacter MoFe protein and clostridium MoFe protein the quadrupole splitting of component D is i n d e p e n d e n t o f t e m p e r a t u x e u p t o 2 0 0 K . F o r c o m p o n e n t F e 2+, t h e values f o r A E Q a r e i n d e p e n d e n t of temperature up to 120 K and decrease to 2.92 mm/s for both azotobacter MoFe and clostridium MoFe p r o t e i n a t 2 0 0 K . F o r c o m p o n e n t M N t h e q u a d ~ u p o l e s p l i t t i n g is i n d e p e n d e n t o f t e m p e r a t u r e u p t o 1 0 0 K ; a t 2 0 0 K , A E Q h a s d e c r e a s e d b y a b o u t 0 . 1 m m / s r e l a t i v e t o its l o w t e m p e r a t u r e v a l u e s . Spectral component
Fe 2+ D S MN M Ox MR
C. p a s t e u r i a n u m
A . vinelandii
Z~SQ ( m m / s )
6 (mm/s)
Total absorption (%)
AEQ (ram/s)
6 (mm/s)
Total absorption (%)
3 . 0 0 (2) 0.70 (2) 1 . 3 7 (5) 0.8 0 . 7 - - 1.1 0.9 -- 1.2
0 . 6 4 (2) 0.64 (2) 0 . 6 4 (5) 0.41 0.35 0.46
1 2 . 8 (5) 39 (2) -~6 42 (2)
3.02 0.61 1.37 0.76 0.8 0.95
0.69 0.64 0.64 0.40 0.37 0.36
1 3 . 0 (5) 42 (2)
(2) (2) (4) (3) -- 1.05 -- 1.3
(2) (2) (4) (3)
40
(2)
has to be between 38--41% of total Fe), and (c) the requirement that the spectrum M1 (see Fig. 6) accounts for 52--55% of the total absorption. The hightemperature results for native clostridium MoFe protein (and for comparison those of azotobacter MoFe protein) are summarized in Table I. To prepare the magnetic spectrum M N we have removed the contributions of D, Fe 2+, and S by subtraction. The results are shown in Fig. 3. (The raw data yielding the spectra in Fig. 3B and C are n o t shown here.) We have evaluated the spectra in Fig. 3 with the spin Hamiltonian
(1)
/4 --/46 + / t N with fie = D[S
+ 1) + X(S
/tN = A0~" 7 - - gn~n~ " Y+/~Q
+
(2) (3)
where
I:IQ - eQ Vzz 1 2 [ 3 ~ -- 15/4 + ~ ( ~ - - ~ ) ]
(4)
All symbols in Eqns. (2)--(4) have their conventional meaning. To evaluate the spectra shown in Fig. 3 we have used the procedures described earlier (see Ref. 5, p. 36, and Ref. 7, p. 197). Assuming 30 Fe atoms for the holoprotein we can infer from Table I that 12 Fe atoms/holoprotein are associated with the cofactor centers. According to an EPR quantitation [ 1 1 ] the protein contains two M N centers. This yields 6 Fe atoms per cofactor center. The rhombicity parameter of the zero-field splitting, k = 0.05, is determined from the observed g values at gy = 4.29, gx = 3.77, and gz = 2.01 (see Figure 1C of Ref. 12 for a
130 TABLE n SPIN H A M I L T O N I A N P A R A M E T E R S U S E D T O G E N E R A T E T H E T H E O R E T I C A L C U R V E S IN F I G . 3 FROM EQNS. 1--4 T h e zaro-field splitting parameter D = +(6 ± 1 . 5 ) c m was d e t e r m i n e d f r o m a series o f high-field m e a s t t r e ment~. Spectral
N u m b e r o f Fe
component
atoms per M center
A1 A2 A3 B
1 1 1 3
A0 gnfl----n( k G )
~ 2
eQVzz
--144 --120 --94 +78
---0.74 ---0.85 +0.84 --0.38
~
8 ( m m / s ) **
(mm/s) *
1.0 0.0 ---0.25 --3.0
0.46 0.46 0.46 0.37
* T h e electric field g r a d i e n t t e n s o r s axe r e f e r r e d t o t h e f r a m e (x, y , z) d e f i n i n g t h e e l e c t r o n i c zero-field splitting. Vzz is n o t necessarily the largest c o m p o n e n t ; ~ = ( V x x - - V y y ) / V z z . ** I s o m e t r i c shift at 4.2 K w i t h r e s p e c t t o m e t a l l i c i r o n at r o o m t e m p e r a t u r e .
representative EPR spectrum). The results of decomposing the spectra into subcomponents A1, A2, A3 and B (see Ilef. 7) are shown in Fig. 3; the parameter set used to compute the theoretical spectra is given in Table II. The following basic assumptions were made for the data evaluation: 1) It is assumed that the M5ssbauer spectrum resulting from the spin-coupled iron atoms of an M-cluster can be computed by summing up the spectra of individual iron sites, all sites sharing a common electronic Hamiltonian, Eqn. (2), but with individual (local) A-tensors. This is strictly true only if the local A-tensors (in the uncoupled representation) are either isotropic or if they share a common principal axis system. 2) The A-tensors in Eqn. (3) are taken to be isotropic. The data are quite sensitive to Ax and Ay, so the sharpness of the absorption lines implies that Ax ~--Ay. The magnetic anisotropy of the +1/2 electronic ground doublet renders the MSssbauer spectrum insensitive to Az. 3) We have assumed that the local electric field gradient tensors share a common principal axis system with the electronic zero-field tensor (which refers to the cluster as a whole). The largest components of the electric field gradient tensors, however, need not be along the electronic z-axis. For further details, such as the selection of the subcomponents and the determination of the zero-field splitting parameter D, the reader is referred to Ref. 7.
Studies under fixing conditions We have studied samples of STFe enriched MoFe protein during turnover with nitrogen as a substrate. Samples prepared by manual mixing and by a rapidmix/rapid quench technique (see Methods) yielded identical MSssbauer spectra. A low-temperature MSssbauer spectrum is shown in Fig. 4A. As reported for azotobacter MoFe protein [5] and klebsiella MoFe protein [ 13], components D and Fe 2÷ appear as observed in the native enzyme (the P-clusters have remained in the state pN). To more closely inspect the spectrum of the cofactor centers, we have subtracted the contributions of components D and Fe z+. The resulting spectrum shown in Fig. 4B is essentially due to the cofactor centers, now observed in the state MR. The shoulder at a velocity of +1.25 mm/s is at the
131 I
I
I
,,P% /'
ill , i
~ 2.G 0
i*
n
'hI
f
rl
,
/~1 lip
0
I i M
2.C
iI I I 1 II tI/ II I I I
i I
,
I
I
I
]
0 2 VKI_OCITY ( mm/s )
,
I 4
Fig. 4. (A) MSssbauer spectrum of clostridium MoFe protein in the presence of Fe-proteiv, MgATP, N2, and dithionite (fixing mixture). The spectrum was taken at 4.2 K in a 600 G parallel field. The spectrum in (B) was obtained by removing the contributions o f D a n d F e 2+ f r o m t h e r a w d a t a . T h e r e s u l t a n t s p e c t x u r n is p r e d o m i n a n t l y due to component M R, the reduced cofactor centers.
same position as the right absorption line of c o m p o n e n t S, suggesting that S is in the same state as observed in the native enzyme. Fig. 4B shows also a broad magnetic c o m p o n e n t (about 5% of total Fe) with absorption stretching from --3 m m / s to +3 mm/s. We have studied an aliquot of the MSssbauer sample with EPR spectroscopy and found that the EPR signal of the S = 3/2 centers had declined to 10% of its original amplitude. Thus the broad magnetic component is the remainder of c o m p o n e n t M N. The absence of any discernable splitting shows that the Cofactor centers have integer or zero electronic spin in the state M R. In order to characterize this state further we have studied the sample in strong applied fields. If S > 0 an applied field will polarize the electronic states giving rise to an internal magnetic field at the nucleus ~/int =--~-~ A/gnfJn, where (~) is an appropriately taken expectation value of the electronic spin (see for instance [14,15]). So for S > 0 the MSssbauer nucleus will sense an effective field J~e~ = ~int + ~ which is different from the applied field, while ~eff = / ~ for a diamagnetic compound. Fig. 5 shows a high-field MSssbauer spectrum of the cofactor centers in the state M a. It was prepared by taking a spectrum at 4.2 K in a parallel field of 55 kG and then subtracting the contributions of components D and Fe 2÷ (these components are diamagnetic and their spectra can be computed with adequate accuracy). The solid line in Fig. 5 is a theoretical spectrum of compoment M R assuming S = 0; the large magnetic splitting (Hin t ~ 100 kG) of the experimental spectrum clearly shows the presence of an internal magnetic field, i.e., S > 0 for the state M R.
132 l.-Z nW 0_ Z Z
o 1.o rn
-4
-2
0 2 VELOCITY ( m m / S )
4
Fig. 5. High-field s p e c t r u m o f t h e c o f a c t o r c e n t e r s in t h e s t a t e M R. T h e d a t a w e r e t a k e n at 4.2 K in a parallel field o f 50 k G . T h e c o n t r i b u t i o n s o f t h e d i a m a g n e t i c c o m p o n e n t s F e 2+ a n d D w e r e r e m o v e d . T h e s o l i d l l n e is a t h e o r e t i c a l s p e c t r u m a s s u m i n g d i a m a g n e t i s m (S = 0 ) for t h e s t a t e MR; it is a p p a r e n t t h a t t h e e l e c t r o n i c g r o u n d m a n i f o l d has S > 0.
Thionine oxidized MoFe protein The addition of thionine (6 e- equivalents per MoFe protein molecule) results in a one-electron oxidation of each cofactor center and each P-cluster. For azotobacter MoFe protein a thionine titration [6] proceeds in two distinct phases: In the first phase, four electrons are utilized to oxidize the four P-clusters from the state pN into the state P°X(E0' ~----250 mV). A second oxidation step, involving two electrons, transforms the cofactor centers into the state M °x. For clostridium MoFe protein the two processes are n o t as well separated presumably because the redox potential of the M°X/M N couple is substantially lower in clostridium MoFe protein [ 16] than in azotobacter MoFe protein. A series of MSssbauer spectra of thionine oxidized clostridium MoFe protein taken at 4.2 K are shown in Fig. 6. We have correlated [6] the components observed in Fig. 6 with those observed for the native enzyme. The central doublet in Fig. 6A results from the cofactor centers in the state M °x. The shape of the high-field spectra shows the absence of an internal magnetic field, i.e. the electronic ground state of M ° x is diamagnetic (S = 0). (We believe [6] that c o m p o n e n t S is also contained in the central doublet of Fig. 6A.) The remainder of the spectrum is a complex magnetic c o m p o n e n t (labeled M1). C o m p o n e n t M1 results from the P-clusters in the state pOX. We have discussed that M1 spectrum of azotobacter MoFe protein in considerable detail (see Appendix of Ref. 6) and we have given a spectral decomposition of the lowfield data (H < 1 kG). In the following we will extend the data analysis to clostridium MoFe protein and include an analysis of the high-field data. Before discussing the high-field spectra we briefly review some of the physics: In our interpretation a P-cluster in the state pOX contains 4 Fe atoms, spin-coupled to yield a system spin S. The spin S is as y e t undetermined, b u t is half integer and restricted to 3/2 ~< S ~< 9/2. The electronic ground manifold can be described by Eqn. 2 with D < 0 and k ~ 0. The lowest Kramers doublet is well separated (by at least 5 cm -1) from the first excited doublet; the ground doublet has a magnetic quantum number m = +S and effective g-values gx gy ~ 0 and gz = 4S (assuming go = 2 in Eqn. 2). A close inspection of the M1
133
2o 1Akc
~
t
4.0 I--
~ o.~ n Z
z 2.C O i.i1_ iT
I
$ : m < oC
2.
-
55
kG
',
,+~t~
i i~
~,
'
,
~1~
I
,
Iii i
I
,
I
-4
I
0
I
,
I
,
I
i
4
VELOCITY (mm/s) Fig. 6. L o w - t e m p e r a t u r e (4.2 K M ~ s s b a u e t s p e c t r a o f t h i o n i n e o x i d i z e d c l o s t r i d i u m M o F e p r o t e i n irL paraliel a p p l i e d fields. T h e c e n t r a l d o u b l e t in t h e 1 k G s p e c t r u m results f r o m t h e s t a t e M O X ; its a b s o r p t i o n p a t t e r n a t h i g h e r field i m p l i e s d i a m a g n e t i s m f o r t h e s t a t e M OX. T h e solid lines are t h e r e s u l t o f a s p e c t r a l d e c o m p o s i t i o n of t h e M1 s p e c t r u m using t h e p a r a m e t e r set in T a b l e I I I . We h a v e m a r k e d w i t h a r r o w s s o m e a b s o r p t i o n lines w h i c h b e l o n g t o c o m p o n e n t s 7 a n d 8 o f T a b l e I I I . T h e s e c o m p o n e n t s are c h a r a c t e r i z e d b y a p o s i t i v e m a g n e t i c h y p e r f i n e c o u p l i n g c o n s t a n t ; thei~ a b s o r p t i o n lines m o v e o u t w a r d ( H e f f i n c r e a s e s ) as t h e s t r e n g t h o f t h e a p p l i e d field increases.
spectrum reveals that it consists of a superposition of eight subspectra, suggesting two sets of slightly inequivalent P-clusters. Previously for azotobacter MoFe protein, we have restricted our data analysis to the low-field spectra, for three reasons: (1) Because k ~ 0 and D < 0 the expectation value of the elctronic spin for the ground doublet is non-zero only along the z
"gnflnHint Iz
(5)
with Hint = "" Ao/gnfln. Thus the magnetic splitting of the MSssbauer spectrum depends on one parameter only. This also holds true if the magnetic hyperfine interaction is described by an A-tensor. (2) The low-field spectra do not depend on the sign of A0. (3) The spectrum resulting from the ground doublet is independent of the magnitude of the zero-field splitting parameter D. A strong applied field could mix the (2S + 1) electronic states of the spin multiplet. This mixing depends on the magnitude of the Zeeman term relative
134
to the zero-field splitting. Appreciable mixing would render the M1 spectrum unmanageably complex because it would produce finite values for (Sx) and (Sy). The direction of ~int relative to the electric field gradient tensor would then depend on the orientation of the molecule relative to the applied field. (If the magnetic hyperfine interaction in Eqn. 3 would be described by an A-tensor one would have to introduce further unknowns.) Level mixing would cause a broadening of the absorption lines of some subcomponents of M1 as the applied field is increased (the details depend on the anisotropy of the A-tensor and on the sign of its components). A close inspection (aided by computer simulations) of the spectra in Fig. 6 (and those taken on azotobacter MoFe protein) reveals that level mixing is negligible for applied fields up to 30 kG and of only minor importance at 55 kG. The observed broadening is almost entirely due to the nuclear Zeeman term in Eqn. 3. If this interpretation is correct one can compute the high-field spectra with the parameter set used to fit the lowfield spectra; the only additional unknowns are the signs of A0 for each subcomponent. We have pointed out earlier [6] that the M1 spectra of clostridium MoFe protein and azotobacter MoFe protein differ only in the MSssbauer parameters of one subcomponent. Thus a complete parameter set for clostridium MoFe protein was available from our azotobacter MoFe protein data analysis. A feature of the azotobacter MoFe protein decomposition was the assumption that half of the eight subcomponents were characterized by a positive hyperfine coupling constant A0. Although only two components with A0 > 0 were clearly evident in the 10 kG and 20 kG spectra, the preliminary analysis of the overall absorption pattern at higher fields suggested to us four components with
TABLE III HYPERFINE PARAMETERS SHOWN IN FIG. 6
AT 4.2 K USED
TO
GENERATE
THE
THEORETICAL
SPECTRA
F o r t h e s i m u l a t i o n s w e u s e d S = 5 / 2 , D = - - 1 0 c m - I , k = 0, a n d gO = 2 in E q n . 2. In t h e f r a m e w o r k of E q n s . 2 a n d 3 t h e i n t e r n a l m a g n e t i c field f o r H > 20 k G is given b y H i n t = +S Ao/gn{3 n, i.e. H i n t h a s the s a m e sign as A 0. T h e p a r a m e t e r a n d t h e E u l e r angles ~, ~ a n d 3, describing the o r e i n t a t i o n o f the local electric field gradient tensors relative to the e l e c t r o n i c s y s t e m are ~soft* p a r a m e t e r s (see A p p e n d i x of R e f . 6). We believe that the signs and m a g n i t u d e s o f H i n t are q u i t e reliable p a r a m e t e r s . A E Q a n d 5 f o r c o m p o n e n t s 7 a n d 8 are d e t e r m i n e d quite precisely f r o m zero-field spectra t a k e n a t 1 4 0 K ; to a c c o u n t f o r the s e c o n d order D o p p l e r shift 0 . 0 5 m m / s was a d d e d t o 5 t o o b t a i n t h e v a l u e s at 4 . 2 K. Q u a d r u p o l e splittings a n d i s o m e r i c shifts o f c o m p o n e n t s 1 - - 6 are less well d e t e r m i n e d . T h e m a g n i t u d e s o f H i n t a n d 5 f o r c o m p o n e n t 4 s e e m to us s o m e w h a t t o o small. T h e essential d i f f e r e n c e b e t w e e n the c l o s t r i d i u m M o F e protein a n d a z o t o b a c t e r M o F e p r o t e i n d a t a is t h e v a l u e of H i n t f o r c o m p o n e n t 2 (Hin t = - - 2 3 7 k G f o r a z o t o b a c t e r M o F e protein). Spectral component
Hint (kG)
AF.Q (mm/s)
6 (ram/s)
"Q
~
1 2 3 4 5 6 7 8
--287 --221 --245 --151 --259 --237 + 201 +223
--1.40 +1.53 +0.57 +0.60 + 1.26 --0.72 + 3.20 +2.30
0.56 0.64 0.40 0.25 0.48 0.49 0.65 0.68
0.70 0.05 0.42 0.32 0.70 0.70 0 0.66
90 0 0 0 0 90 0 0
,6
'T
--.90 18 62 4 58 --90 77 75
61 90 0 0 0 69 90 90
135
A0 > 0. After a systematic analysis of the azotobacter MoFe protein and clostridium MoFe protein high-field data we are now forced to the conclusion that only two subcomponents have A0 > 0. High-filed studies at T = 140 K have shown [7] that the iron sites which yield doublets I and II (the oxidized form of native component Fe 2÷) have A0 > 0. Thus the low-temperature components with A0 > 0 must have values for Z~EQand ~ which characterize doublets I and II. This assignment required a repartitioning of four subcomponents (3, 4, 7 and 8 of Table III in Ref. 6). We found a new spectral decomposition that fits the whole data set on clostridium MoFe protein and azotobacter MoFe protein remarkably well. In particular it properly describes the complex pattern of outward and inward moving absorption lines observed at higher fields. The solid lines in Fig. 6 are theoretical spectra computed from Eqns. 1--4 with the parameter set given in Table III. We have used S = 5/2 for the unknown electronic spin; as long as level mixing is negligible the particular value used for S is unimportant. We believe that our model of the P-clusters (D < 0, ~ ~ 0, four iron sites per P-cluster, two inequivalent sets in the state pO x) properly explains the relevant features of the observed spectra over a wide range of experimental conditions. It is virtually impossible to assign meaningful uncertainties to the parameters quoted in Table III. We have, however, tried to assess the situation by separat~ ing what we consider the more reliable parameters from the 'softer' ones. The softness of parameters is primarily related to the fundamental ambiguity problem discussed in Ref. 6. The parameter set quoted in Table III should be a useful reference for researchers studying nitrogenase from other organisms. Discussion The MSssbauer data obtained for azotobacterMoFe protein and clostridium MoFe protein are strikingly similar, suggesting practically identical metal centers in both proteins *. Our studies have led to a remarkably consistent picture; the holoenzymes (Mr 220 000--250 000) contains two M-centers and four P-clusters accounting for 28 of 30 Fe atoms. The cofactor centers M are novel structures which have been stabilized in three oxidation states. The state MN, observed for the protein as isolated, has electronic spin S = 3/2. All properties connected with an S = 3/2 spin system have been observed with both EPR and MSssbauer spectroscopy: The prominent EPR signals around g = 4.3, 3.7, and 2.0 and the MSssbauer spectra of Fig. 3 result from the m = +1/2 ground doublet. An EPR signal at g = 5.85 [11] and some MSssbauer absorption lines (for clostridium MoFe protein, Huynh, B.H. and Miinck, E., unpublished data) have also been observed for the
* This s t a t e m e n t refers t o t h e structural p r o p e r t i e s o f t h e c e n t e r s as e l i c i t e d b y E P R a n d M ~ s s b a u e r s p e c t r o s c o p y . O n o t h e r levels p r o n o u n c e d d i f f e r e n c e s b e t w e e n t h e p r o t e i n s have b e e n r e p o r t e d . F o r i n s t a n c e , t h e P o t e n t i a l o f t h e M O X / M N c o u p l e is 1 4 0 m V l o w e r in klebsiella Mol~e p r o t e i n t h a n in a z o t o b a c t e r M o F e p r o t e i n [ 1 6 ] . A l s o c r o s s - r e a c t i o n s b e t w e e n F e p r o t e i n s and M o F e p r o t e i n s [ 1 7 ] r e v e a l p a t t e r n s w h i c h s e e m t o r e f l e c t t h e d o m i n a t i n g i n f l u e n c e o f t h e p r o t e i n s t r u c t u r e rather t h a n s t r u c t u r a l features of the MoFe protein metal centers.
136 m = +3/2 excited state doublet. Both techniques have yielded, within the uncertainties, the same value for the zero-field splitting parameter D = +(6 + 1.5) cm -1 for both azotobacter MoFe protein and clostridium MoFe protein. The spin S = 3/2, which refers to the cluster as a whole, results from spincoupling of (most likely) six Fe atoms and (presumably) one Mo atom. Spincoupling is proven by the fact that the spin is shared by six iron atoms and by the observation of positive and negative hyperfine coupling constants. (Positive coupling constants result when the individual spin is oriented antiparallel to the system spin.) Although all iron atoms o f the M-centers have nearly the same quadrupole splitting AEq they are quite inequivalent when their magnetic properties are examined; the magnitudes of Ao/gn~n range from 75 kG to 145 kG. Extended X-ray absorption fine structure data [18] implicate m o l y b d e n u m as a structural c o m p o n e n t of the M-centers. Experiments with samples enriched in 9SMo have failed to show any broadening of the EPR lines due to hyperfine interactions of the 9SMo nucleus with the electronic spin of the M-center. These results are somewhat disturbing; however, they constitute no evidence against the presence of m o l y b d e n u m in the M-centers, since peculiarities of the spincoupling mechanism or 9SMo quadrupolar effects could be at the r o o t of this problem. One-electron redox reactions yield the states M °x (S = 0) and M a (S > 0, integer). The quadrupole doublets observed in the states M a and M °x are n o t as sharp as that observed in the state M N, implying that the individual subsites are somewhat inequivalent (see Table I). The (average) isomeric shift increases by a b o u t 0.06 mm/s per electron added to the cluster. For comparison, the average isomeric shift of 4Fe-4S centers changes by a b o u t 0.15 mm/s per one-electron redox step [19]. In the native enzyme and under fixing conditions the P-clusters are observed in the state pN. A P-cluster in this oxidation state contains three irons of the D-type and one iron site yielding c o m p o n e n t Fe :÷. W e have studied both MSssbauer components in azotobacter MoFe protein and clostridium MoFe protein in applied fields up to 55 kG in the temperature range from 1.5 to 200 K. These studies firmly establish the diamagnetism of the state pN. To within +0.02 mm/s all 12 D-irons of azotobacter MoFe protein exhibit a quadrupole splitting AEQ = 0.81 mm/s. Curiously, all D-irons of clostridium MoFe protein have, within the same narrow margins, AEQ = 0.70 mm/s. Thus the environments of t h e P-clusters differ in both proteins such that all D-irons are affected in the same way. A one-electron oxidation transforms the P-clusters into the state pOX which has half-integer electronic spin 3/2 ~< S ~< 9/2. It is n o t e w o r t h y that the state pOX is EPR-sflent for all proteins investigated so far. We have suggested [6] that a large and negative zero-field splitting parameter D, in conjunction with ), ~ 0, together with fast relaxation for T > 10 K is responsible for this observation. This suggestion is supported by the present study: The analysis of the high-field data suggests a large value for D. For instance, for S = 5/2 our analysis yields [DI> 5 cm-*. For D = - - 5 cm -1 the EPR-active state (which would yield resonances around g = 6 and g = 2) would be 6D -- 30 cm -1 ~ 42 K above the ground state and therefore only marginally populated at T < 10 K. The
137
electronic spin S is as yet undetermined *. Using the information obtained from the M5ssbauer studies the magnetic susceptibility, for T <: 6 K, is predicted to be entirely due to the lowest Kramers doublet and is given by X = S2{J2/kT per P-cluster (assuming go = 2 in Eqn. 2 and H < < kT). Thus lowtemperature susceptibility studies on thionine oxidized MoFe protein (the state M°x is diamagnetic) could be used to determined the unknown spin. The high-field MSssbauer studies and the quantitation of the azotobacter MoFe protein thionine titrations [6] prove independently that the P-clusters contain spin-coupled iron atoms. The analysis of the high-field MSssbauer data shows that one iron site of a P-cluster (the site yielding M5ssbauer component Fe 2÷) has a positive A0 while the other three are characterized by A0 < 0. In contrast, the oxidized high-potential-iron-protein from Chromatium vinosum and the reduced 4Fe-4S Bacillus stearothermophilus ferredoxin apparently have two iron atoms each with positive and negative A0 [19]. Component S is present in equal amounts (about 6% of the total Fe, or 2Fe atoms) in azotobacter MoFe protein, clostridium MoFe protein, and apparently also in klebsiella MoFe protein with practically identical M5ssbauer parameters (AEQ ~-- 1.35 mm/s and 8 = 0.60 ram/s). The MSssbauer parameters are quite atypical of commonly observed impurities (high-spin ferric or ferrous ions). Also no EPR signal attributable to S has been observed despite the fact that samples have been studied over a wide range of redox conditions. The iron atoms of component S are certainly not a structural part of the cofactor centers. This is proven by the fact that the component S (or a component with similar isomeric shift) is absent in isolated cofactor material [11]. It is also quite unlikely, although not strictly ruled out, that S is a structural component of the P-clusters; in that case, however, the M1 spectrum would have to account for 60% of the total absorption, in contrast to the experimental results (52--55%). A further elucidation of the nature of component S will be achieved when reconstituted MoFe protein preparations with selective STFe enrichment for the M- or P-clusters become available. Acknowledgements This work was supported by the National Science Foundation grant PCM08522, by the National Institutes of Health through grant GM17170, by the Graduate Research Committee of the University of Wisconsin, and by Research Career Development Award K04-GM70683 (E.M.). W.H.O-J. is an I.H. Romnes Faculty Fellow of the University of Wisconsin. References 1 O n - h e - J o h n s o n , W . H . , H a m i l t o n , W . D . , L j o n e s , T., Tso, M.-Y.W., B u r r i s , R.H.~ S h a h , V . K . a n d Brill, W . J . ( 1 9 7 2 ) P r o c . N a t l . A c a d . Sci. U . S . A . 6 9 , 3 1 4 2 - - 3 1 4 5 * We ha~e o b s e r v e d r e p r o d u c i b l y a w e a k , t r a n s i e n t E P R signal w i t h g-values a r o u n d 6 . 3 a n d 5 . 6 w h i c h results f r o m a n S = 5 / 2 s y s t e m w i t h k ~ 0 . 0 2 . T h e t e m p e r a t u r e d e p e n d e n c e o f the signal establishes f i r m l y t h a t it r e s u l t s f r o m an e x c i t e d electronic state (thus e x c l u d i n g a h e m e i m p u r i t y ) , s e p a r a t e d i n energy b y a b o u t 3 0 c m - I f r o m t h e g r o u n d s t a t e . T h u s t h e s i g n a l r e s u l t s f r o m a n e l e c t r o n i c s y s t e m w i t h the s a m e properties as that o f t h e state p O X ( E m p t a g e , M., O r m e * J o h n s o n , W . H . , H u y n h , B . H . , a n d M i l n c k , E., u n p u b l i s h e d r e s u l t s ) .
138 2 Zumft, W.G. and Mortenson, L.E. (1975) Biochim. Biophys. Acta 416, 1--55 3 Winter, H.C. and Burrls, R.H. (1976) Annu. Rev. Biochem. 45, 409--426 4 0 r m e - J o h n s o n , W.H., Davis, L.C., Henzl, M.T., Avernl, B.A., Orme-Johnson, N.R., Miinck, E. and Zimmermann, R. (1977) in Recent Developments in Nitrogen Fixation (Newton, W., Postgate, J.R. and Rodriguez-Barrueco, C., eds.), pp. 131--178, Academic Press, New York 5 Milnck, E., Rhodes, H., Orme-Johnson, W.H., Davis, L.C., Brill, W.J. and Shah, V.K. (1975) Biochim. Biophys. Acta 400, 32--53 6 Zimmernann, R., Miinck, E., Brill, W.J., Shah, V.K., Henzl, M.T., Rawlings, J. and Orme-Johnson, W.H. (1978) Biochim. Biophys. Acta 537, 185--207 7 Huynh, B.H, M(inck, E. and Orme-Johnson, W.H. (1979) Biochirn. Biophys. Acta 527, 192--203 8 Zumft, W.G., Mortenson, L.E. and Palmer, G. (1974) Eur, J. Biochem. 46, 525--535 9 Donald, C., Passey, B.I. and Swaby, R.J. (1952) J. Gen. Microbiol. 7, 211--220 10 Emptage, M.H., Zimmermann, R., Que, L., Mflnck, E., Hamilton, W.D. and Orme-Johnson, W.H. (1977) Biochim. Biophys. Acta 495, 12--23 11 Rawlings, J., Shah, V.K., Chisneil, J.R., BI"JlI, W.J., Zimmermann, R., Milnck, E. and Orme-Johnson, W.H. (1978) J. Biol. Chem. 253, 1001--1004 12 Orme-Johnson, W.H. and Davis, L.C. (1977) in Iron-Sulfur Proteins (Lovenberg, W., ed.), Vol. 3, pp. 15--60, Academic Press, New York 13 Smith, B.E. and Lang, G. (1974) Biochem. J. 137, 169--180 14 Milnck, E. (1978) in The Porphyrins (Dolphin, D., ed.), Vol. IV, Chapter 8, pp. 379---423, Academic Press, New York 15 Lang, G. (1970) Q. Rev. Biophys. 3, 1--60 16 O'Donnell, M.J. and Smith, B.E. (1978) Biochem. J. 137, 831--839 17 Emmerich, D.W. and Burris, R.H. (1978) J. Bacteriol. 134,936--943 18 Cramer, S.P., Hodgson, K.O., GUlum, W.O. and Mortenson, L.E. (1978) J. Am. Chem. Soc. 100, 3398--3407 19 C a m m a c k , R., Dickson, D.P.E. and Johnson, C.E. (1977) in Iron-Sulfur Proteins (Lovenberg, W., ed.), Irol. 3, pp. 283--330, Academic Press, New York
Biochimica et Biophysica Acta, 623 (1980) 124--138
© Elsevier/North-Holland Biomedical Press
BBA 3 8 3 9 4 NITROGENASE XII MOSSBAUER STUDIES OF THE MoFe PROTEIN FROM C L O S T R I D I U M P A S T E U R I A N U M W5 B.H. HUYNH a, M.T. HENZL b, J.A. CHRISTNER a, R. ZIMMERMANN c, W.H. ORME-JOHNSON b and E. MUNCK a a Gray Freshwater Biological Institute/Department of Biochemistry, University of Minnesota, Navarre, MN 55392, b Department of Biochemistry, Center for Studies of Nitrogen Fixation, College of Agricultural and Life Sciences, University of Wisconsin, Madison, WI 53706 (U.S.A.) and c Physikalisches Institut, University of Erlangen, D-8520 Erlangen (F.R.G.)
(Received September llth, 1979) Key words: Nitrogenase; MSssbauer spectroscopy; MoFe protein; (C. pasteurianum)
Summary We have studied the molybdenum-protein (MoFe protein) from Clostriwith MSssbauer spectroscopy in the temperature range from 1.5 to 200 K in magnetic fields up to 55 kG. Except for some small differences in the hyperfine parameters the results for the C. p a s t e u r i a n u m prorein are essentially the same as those published previously for the protein from A z o t o b a c t e r vinelandii, i.e. (30 + 2) Fe atoms partition into t w o identical cofactor centers M (each center most likely containing six Fe atoms and one Mo atom), four P-clusters (each center containing four Fe atoms), and one iron environment labeled S (about t w o Fe atoms per holoenzyme). We have analyzed the spectra of the cofactor centers in three distinct oxidation states, dium pasteurianum
e--
e-
M O X ~ MN - - , MR . The diamagnetic (electronic spin S = 0) state M ° x is attained by oxidation of the native, EPR-active (S = 3/2) state M N. The reduce~t state M R is observed in steady state under nitrogen fixing conditions; high-field MSssbauer studies show that the cofactor centers are paramagnetic (integer electronic spin S i> 1) in the state M R. We have evaluated the complex high-field spectra resulting from the P-clusters in the oxidized state pOX. The analysis shows that one iron site is characterized by a positive hyperfine coupling constant A0 while the other three sites have A0 < 0. A slightly modified set of parameters also fits the high-field data of the MoFe protein from A. vinelandii. Finally, we will present a discussion summarizing our principle results obtained to date for the proteins from A. vinelandii and C. p a s t e u r i a n u m .
125 Introduction
A current and widely held working hypothesis [1--4] about the mechanism of nitrogenase is that MgATP hydrolysis-driven electron transfer from the Fe protein leads to reduced redox centers in the MoFe protein. These are subsequently reoxidized during the reduction of nitrogenase by substrates such as H ÷, C2H2 and N2. In parts VIII [5], X [6], and XI [7] of this series we have examined the MoFe protein from Azotobacter vinelandii, using combined EPt~ and MSssbauer spectroscopy, in an effort to characterize the redox centers in the protein. We believe that in all essential respects MoFe protein from any source will be found to have the same catalytic groups; we have previously commented [ 5,6] on the similarity of MSssbauer spectra from Klebsiella pneumoniae and A. vfnelandii MoFe proteins. As part of a limited survey of the physical properties of nitrogenases, and because the Clostridium pasteurianum enzyme has played a central role in the development of the biophysical picture of nitrogenase [ 1,8], we undertook to study this protein in some detail. Materials and Methods C. pasteurianum W5 was grown on a nitrogen-free medium in 18 1 cultures. The medium contained (per 1): 20 g sucrose, 0.1 M potassium phosphate (pH 7.6), 20 mM potassium citrate, 10 mg NaC1, 50mg CaC12 • 2 H20, 0.5 g MgSO4 • 7 H20, 20 mg Na2MoO4 • 2 H20, 1 mg STFe, and 50 Izg biotin. The potassium phosphate was added as a 1 M solution prepared from reagent grade KOH and H3PO4. Contaminating S6Fe was removed from the sucrose and phosphate solu. tions by autoclaving them in the presence of A1203 [9] and filtering after they had been allowed to stand for several days at 4°C. The doubling time for the organism at 30°C on this medium was 120 min. Nitrogen was bubbled through the culture at 2--3 1/min. When the absorbance of the culture (measured at 650 nm in a 1 cm pathlength cuvette on an aliquot of the culture, appropriately diluted such that the spectrophotometer reading was less than 0.4) reached 1.8, the culture was harvested with the aid of a small Sharples centrifuge. The cell paste (50--75 g) was immediately frozen in liquid nitrogen. Nitrogenase was purified as follows: Aerobic buffer (25 mM Tris-HC1, pH 8.5, at 30°C) was added to frozen C. pasteurianum cell paste in a weight ratio 3 : 1 . Lysosyme (1 mg/cell paste) and deoxyribonuclease I (0.1 mg/g cell paste), both from Worthington, were added. The resulting suspension was shaken under hydrogen for 1 h at 30°C. Cell debris was removed by centrifuga. tion (11 000 × g) for 45 rain. After equilibrium the supernatant with CO (0.2 atom in a sealed suction flask) and adding dithionide, it was loaded onto an anaerobic column of DEAE-cellulose (Whatman DE32). After washing the column with 0.15 NaC1 the MoFe protein and the Fe protein were eluted with 0.25 and 0.4 M NaC1, respectively. Both solutions were then equilibrated with CO, which serves to inhibit chlostrial hydrogenase. All chromatographic steps ~n the isolation were preformed at 15 ° C. The clostridium MoFe protein fractions was further purified by chromatography on Sephadex 6B (Pharmacia), equilibrated with 50 mM Tris-HC1, pH 8.0 (30°C). The fractions of highest specific activity were then concentrated and
126
subjected to chromatography on DEAE-cellulose, employing a NaC1 gradient (0.15 M--0.30 M) for elution. The most active fractions were combined and concentrated. The Fe protein fraction was further purified by chromatography on Sephadex G-100 (Pharmacia), equilibrated with 50 mM Tris-HC1, pH 8.0, containing 20% (v/v) glycerol. The STFe-enriched MoFe protein preparation used in these experiments had a protein concentration of 37 mg/ml, reduced 1200 nmol C2H2 per min per mg, and contained 22 gatoms of Fe per mol. The Fe protein solution contained 14 mg protein per ml and had a specific activity of 2200 nmol • min-' • mg-'.
Nitrogenase during turnover -- preparation o f MiSssbauer samples (a) Manual mixing. 0.58 ml clostridium MoFe protein (97 nmol) was combined with 0.42 ml clostridium Fe protein (103 nmol) in a N2-filled serum bottle. 0.025 ml of 1 M Na2S204 in 0.1 M Tris base was added, and the solution was incubated for 5 min at 0°C. Then 0.2 ml of cold (0°C) anaerobic MgATP generator was introduced by syringe. 30 s after adding the generator, the reaction mixture was injected into a MSssbauer cuvette, housed in a N2-filled loading chamber which had been pre-cooled with liquid nitrogen. The sample was frozen within 2 min after initiating the reaction. In a control experiment monitored with EPR spectroscopy, we demonstrated that, at 0°C, the height of the g = 3.78 feature of the MoFe protein spectrum had decreased by 87% after 30 s of reaction. The MgATP generating system was prepared by dissolving 120 mg disodium A T P - 3 H20 (P-L Biochemicals) and 530 mg creatine phosphate (Pierce Chemical Co.) in 1 ml of glass-distilled water, and the pH was adjusted to 7. Then 15 mg of creatine phosphokinase (Sigma) and 300 pl of 1 M Mg(CH3COO)2 • 4 H20 were added, and the volume was adjusted to 2.0 ml. (b) Rapid mixing. 0.30 ml of clostridium MoFe protein was rapidly mixed with 0.15 ml of clostridium Fe protein to give final concentrations of 120 and 80 #M, respectively, for the two proteins. The syringe containing the clostridium Fe protein solution also contained Mg2÷, disodium ATP • 3 H20, creatine phosphate, and creatine phosphokinase at levels such that, after mixing the following concentrations were achieved: 2.5 mM Mg2÷, 2.5 mM ATP, 75 mM creatine phosphate, 13 mg/ml creatine phosphokinase (1900 I.U./ml). After incubation at 30°C for 8 s, the reaction mixture was shot into cold (120 K) isopentane. After the frozen material had settled, it was packed into a MSssbauer cuvette. A parallel sample collected for examination by EPR spectroscopy revealed that the height of the g = 3.78 signal had decreased by at least 90%. The Mbssbauer spectrometer at the Gray Freshwater Biological Institute and the methodology of Mbssbauer data analyses have been described previously [6,10]. Results
For background information about the P-clusters and the M-centers (cofactor centers) the reader is referred to the literature [5--7,11]. Fig. 1 summarizes the various oxidation states which have been attained and which we have characterized for azotobacter MoFe protein. The nomenclature of the MSssbauer components is given in Table I of Ref. 6.
127 i
O.0 4.2K
I
.........' ",,'
I--- 3 . 0 Z bJ U tY W
COFACTOR
CENTER M
M"
M ox
MR
I
z__o.o .................................... z_o
I
,:............... ,' ,%
, , ,,,' , , /
Fe"
_ ..........2
i---
e-
e-..
S=3~
S=O
~ m 3£ S~1
(EPR active) F-
,
""
P- CLUSTER 27K
pOX
"%",
.....",,, ,,,,,:
,,,'
p.
e-.
~, --~--->,Fe '÷
I
/ .D--/-;S
S_>~ (EPR silent )
S=O
I
-2
i
0 2 VELOCITY ( m m / s )
,
I
4
Fig. 1. S t a b l e o x i d a t i o n s t a t e s o f t h e c o f a c t o r c e n t e r s a n d t h e P - c l u s t e r s . T h e c u b a n e 4 F e - 4 S s t r u c t u r e d e p i c t e d f o r t h e P - c l u s t e r is v e r y p r o b a b l e , b u t n o t a n e s t a b l i s h e d f a c t . T h e l e t t e r s D s y m b o l i z e t h r e e i r o n a t o m s w h i c h give rise t o t h e q u a d r u p o l e d o u b l e t l a b e l e d D ; t h e F e 2+ site y i e l d s t h e d o u b l e t l a b e l e d F e 2+. Fig. 2. M 6 s s b a u e r s p e c t r a o f n a t i v e M o F e p r o t e i n f r o m C. pasteurianurn W 5 t a k e n in a p a r a l l e l field o f 6 0 0 G. T h e s p e c t r a i n ( A ) a n d (B) w e r e o b t a i n e d a t 4 . 2 K a n d 7 0 K , r e s p e c t i v e l y . T h e s o l i d Hne i n (B) is the result of fitting four doublets to the data with the constraints described in the text. The deconvoluted s p e c t r u m i n (C) w a s o b t a i n e d as d i s c u s s e d f o r t h e a z o t o b a c t e r M o F e p r o t e i n [ 7 ] .
Results for the native MoFe protein Fig. 2A shows a spectrum of STFe enriched clostridium MoFe protein taken at 4.2 K in a parallel field of 600 G. (We have recorded approx. 150 spectra of t w o clostridium MoFe protein batches in the temperature range from 1.5 to 240 K in fields up to 55 kG). Fig. 2B shows a spectrum of the same sample taken at 70 K. In Fig. 2 the t w o quadrupole doublets D and Fe 2÷ (the signature of the P-clusters) are clearly discerned. C o m p o n e n t M N, the spectrum resulting from the cofactor centers in their EPR active state, appears at 4.2 K as a magnetic spectrum (shown in Fig. 3 below). A t 70 K the electronic spin S = 3/2 of the cofactor centers relaxes rapidly; the magnetic hyperfine interactions are averaged o u t and c o m p o n e n t M N appears as a quadrupole d o u b l e t in Fig. 2B. To re¢olve c o m p o n e n t S optimally we have used the same procedure as described for azotobacter MoFe protein [7]: the spectrum was recorded at a temperature (27 K) where c o m p o n e n t M N is strongly broadened by relaxation, and then deconvoluted. The deconvoluted spectrum demonstrates the presence
128
of S and it defines the line positions and widths of the doublets, D, Fe 2÷, and S quite precisely. The shape of c o m p o n e n t M N at 70 K was obtained by subtracting the spectrum of Fig. 2B from that of Fig. 2A. Since the MSssbauer parameters of D, Fe 2÷ and S are independent of temperature for T < 100 K the contributions of these species cancel. The resulting difference spectrum is the magnetic spectrum M s superimposed with an inverted doublet, the M s spectrum at 70 K. The lineshape of M E is somewhat non-Lorentzian (12 Fe/molecule contribute!) and has a full width at half max. of a b o u t 0.33 mm/s. For the (average) quadrupole splitting and isomeric shift we found AEQ = 0.81 mm/s a n d ~ = 0.41 mm/s (with respect to metallic Fe), respectively. With a precise knowledge of all line positions and good information a b o u t their widths we have least-squares fitted the spectrum in Fig. 2B assuming four quadrupole doublets. This procedure yields a reliable value for the fraction at the Fe 2÷ sites (12.8%), in agreement with the results on azotobacter MoFe protein (13%). The relative intensities of the D and M s doublets could n o t be reliably determined with this procedure primarily because M s has somewhat non-Lorentzian lines. We have therefore fixed the intensity of doublet D by assuming that the D and Fe 2÷ sites occur in the ratio of 3 : 1. This yields an intensity for D consistent with (a) the results on azotobacter MoFe protein, (b) the requirements for preparing the spectra of Fig. 3 (the 4.2 K spectra show that the absorption of D
0.0 0.~
'
I
'
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0 . 6 kG
i
\
I
,
'
~
I..,nr
m
<
-4
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U L V E L O C I T Y (m rn/s )
"*
Fig. 3. L o w - t e m p e r a t u r e M ~ s s b a u e r s p e c t r a o f the c o f a c t o r c e n t e r s M i n 6 0 0 G p a r a l l e l (A), 6 0 0 G t r a n s verse (B), a n d 5 0 k G p a r a l l e l (C) m a g n e t i c fields. T h e s p e c t r a w e r e o b t a i n e d f r o m t h e r a w d a t a b y r e m o v i n g the c o n t r i b u t i o n s o f D , F e 2+, a n d S. B e c a u s e o f the large c o n t r i b u t i o n o f c o m p o n e n t D the shapes o f t h e 'experimental' spectra in ( A ) a n d (B) a r e n o t v e r y well d e f i n e d in t h e v e l o c i t y r a n g e f r o m - - 0 . 5 m m / s t o + 1 . 5 m m / s . T h e s o l i d lines a r e t h e o r e t i c a l spectra generated w i t h the parameter set listed in T a b l e II. A l m o s t i d e n t i c a l t h e o r e t i c a l s p e c t r a o f the s u b c o m p o n e n t s A 1 , A 2 , A 3 , a n d B ( t h r e e i d e n t i c a l s i t e s ) h a v e b e e n p l o t t e d previously for a z o t o b a c t e r M o F e p r o t e i n [ 7 ] . T h e a g r e e m e n t b e t w e e n e x p e r i m e n t a n d t h e o r y i n C c o u l d be i m p r o v e d b y a l l o w i n g d i f f e r e n t h y p e r f i n e p a r a m e t e r s for the three B - c o m p o n e n t s .
129 TABLE I M{'~SSBAUER P A R A M E T E R S PROTEIN
FOR
CLOSTRIDIUM
MoFe PROTEIN
AND AZOTOBACTER
MoFe
The data on azotobacter MoFe protein are taken from Ref. 5 and 7. Isomeric shifts are quoted at 4.2 K relative t o m e t a l l i c i r o n a t r o o m t e m p e r a t u r e . T h e n u m b e r s in p a r e n t h e s e s give t h e e r r o r in t h e l a s t signific a n t digit. If n o u n c e r t a i n t i e s a r e q u o t e d , t h e p a r a m e t e r s a r e a v e r a g e v a l u e s o f u n r e s o l v e d s u b e o m p o n e n t s . For both azotobacter MoFe protein and clostridium MoFe protein the quadrupole splitting of component D is i n d e p e n d e n t o f t e m p e r a t u x e u p t o 2 0 0 K . F o r c o m p o n e n t F e 2+, t h e values f o r A E Q a r e i n d e p e n d e n t of temperature up to 120 K and decrease to 2.92 mm/s for both azotobacter MoFe and clostridium MoFe p r o t e i n a t 2 0 0 K . F o r c o m p o n e n t M N t h e q u a d ~ u p o l e s p l i t t i n g is i n d e p e n d e n t o f t e m p e r a t u r e u p t o 1 0 0 K ; a t 2 0 0 K , A E Q h a s d e c r e a s e d b y a b o u t 0 . 1 m m / s r e l a t i v e t o its l o w t e m p e r a t u r e v a l u e s . Spectral component
Fe 2+ D S MN M Ox MR
C. p a s t e u r i a n u m
A . vinelandii
Z~SQ ( m m / s )
6 (mm/s)
Total absorption (%)
AEQ (ram/s)
6 (mm/s)
Total absorption (%)
3 . 0 0 (2) 0.70 (2) 1 . 3 7 (5) 0.8 0 . 7 - - 1.1 0.9 -- 1.2
0 . 6 4 (2) 0.64 (2) 0 . 6 4 (5) 0.41 0.35 0.46
1 2 . 8 (5) 39 (2) -~6 42 (2)
3.02 0.61 1.37 0.76 0.8 0.95
0.69 0.64 0.64 0.40 0.37 0.36
1 3 . 0 (5) 42 (2)
(2) (2) (4) (3) -- 1.05 -- 1.3
(2) (2) (4) (3)
40
(2)
has to be between 38--41% of total Fe), and (c) the requirement that the spectrum M1 (see Fig. 6) accounts for 52--55% of the total absorption. The hightemperature results for native clostridium MoFe protein (and for comparison those of azotobacter MoFe protein) are summarized in Table I. To prepare the magnetic spectrum M N we have removed the contributions of D, Fe 2+, and S by subtraction. The results are shown in Fig. 3. (The raw data yielding the spectra in Fig. 3B and C are n o t shown here.) We have evaluated the spectra in Fig. 3 with the spin Hamiltonian
(1)
/4 --/46 + / t N with fie = D[S
+ 1) + X(S
/tN = A0~" 7 - - gn~n~ " Y+/~Q
+
(2) (3)
where
I:IQ - eQ Vzz 1 2 [ 3 ~ -- 15/4 + ~ ( ~ - - ~ ) ]
(4)
All symbols in Eqns. (2)--(4) have their conventional meaning. To evaluate the spectra shown in Fig. 3 we have used the procedures described earlier (see Ref. 5, p. 36, and Ref. 7, p. 197). Assuming 30 Fe atoms for the holoprotein we can infer from Table I that 12 Fe atoms/holoprotein are associated with the cofactor centers. According to an EPR quantitation [ 1 1 ] the protein contains two M N centers. This yields 6 Fe atoms per cofactor center. The rhombicity parameter of the zero-field splitting, k = 0.05, is determined from the observed g values at gy = 4.29, gx = 3.77, and gz = 2.01 (see Figure 1C of Ref. 12 for a
130 TABLE n SPIN H A M I L T O N I A N P A R A M E T E R S U S E D T O G E N E R A T E T H E T H E O R E T I C A L C U R V E S IN F I G . 3 FROM EQNS. 1--4 T h e zaro-field splitting parameter D = +(6 ± 1 . 5 ) c m was d e t e r m i n e d f r o m a series o f high-field m e a s t t r e ment~. Spectral
N u m b e r o f Fe
component
atoms per M center
A1 A2 A3 B
1 1 1 3
A0 gnfl----n( k G )
~ 2
eQVzz
--144 --120 --94 +78
---0.74 ---0.85 +0.84 --0.38
~
8 ( m m / s ) **
(mm/s) *
1.0 0.0 ---0.25 --3.0
0.46 0.46 0.46 0.37
* T h e electric field g r a d i e n t t e n s o r s axe r e f e r r e d t o t h e f r a m e (x, y , z) d e f i n i n g t h e e l e c t r o n i c zero-field splitting. Vzz is n o t necessarily the largest c o m p o n e n t ; ~ = ( V x x - - V y y ) / V z z . ** I s o m e t r i c shift at 4.2 K w i t h r e s p e c t t o m e t a l l i c i r o n at r o o m t e m p e r a t u r e .
representative EPR spectrum). The results of decomposing the spectra into subcomponents A1, A2, A3 and B (see Ilef. 7) are shown in Fig. 3; the parameter set used to compute the theoretical spectra is given in Table II. The following basic assumptions were made for the data evaluation: 1) It is assumed that the M5ssbauer spectrum resulting from the spin-coupled iron atoms of an M-cluster can be computed by summing up the spectra of individual iron sites, all sites sharing a common electronic Hamiltonian, Eqn. (2), but with individual (local) A-tensors. This is strictly true only if the local A-tensors (in the uncoupled representation) are either isotropic or if they share a common principal axis system. 2) The A-tensors in Eqn. (3) are taken to be isotropic. The data are quite sensitive to Ax and Ay, so the sharpness of the absorption lines implies that Ax ~--Ay. The magnetic anisotropy of the +1/2 electronic ground doublet renders the MSssbauer spectrum insensitive to Az. 3) We have assumed that the local electric field gradient tensors share a common principal axis system with the electronic zero-field tensor (which refers to the cluster as a whole). The largest components of the electric field gradient tensors, however, need not be along the electronic z-axis. For further details, such as the selection of the subcomponents and the determination of the zero-field splitting parameter D, the reader is referred to Ref. 7.
Studies under fixing conditions We have studied samples of STFe enriched MoFe protein during turnover with nitrogen as a substrate. Samples prepared by manual mixing and by a rapidmix/rapid quench technique (see Methods) yielded identical MSssbauer spectra. A low-temperature MSssbauer spectrum is shown in Fig. 4A. As reported for azotobacter MoFe protein [5] and klebsiella MoFe protein [ 13], components D and Fe 2÷ appear as observed in the native enzyme (the P-clusters have remained in the state pN). To more closely inspect the spectrum of the cofactor centers, we have subtracted the contributions of components D and Fe z+. The resulting spectrum shown in Fig. 4B is essentially due to the cofactor centers, now observed in the state MR. The shoulder at a velocity of +1.25 mm/s is at the
131 I
I
I
,,P% /'
ill , i
~ 2.G 0
i*
n
'hI
f
rl
,
/~1 lip
0
I i M
2.C
iI I I 1 II tI/ II I I I
i I
,
I
I
I
]
0 2 VKI_OCITY ( mm/s )
,
I 4
Fig. 4. (A) MSssbauer spectrum of clostridium MoFe protein in the presence of Fe-proteiv, MgATP, N2, and dithionite (fixing mixture). The spectrum was taken at 4.2 K in a 600 G parallel field. The spectrum in (B) was obtained by removing the contributions o f D a n d F e 2+ f r o m t h e r a w d a t a . T h e r e s u l t a n t s p e c t x u r n is p r e d o m i n a n t l y due to component M R, the reduced cofactor centers.
same position as the right absorption line of c o m p o n e n t S, suggesting that S is in the same state as observed in the native enzyme. Fig. 4B shows also a broad magnetic c o m p o n e n t (about 5% of total Fe) with absorption stretching from --3 m m / s to +3 mm/s. We have studied an aliquot of the MSssbauer sample with EPR spectroscopy and found that the EPR signal of the S = 3/2 centers had declined to 10% of its original amplitude. Thus the broad magnetic component is the remainder of c o m p o n e n t M N. The absence of any discernable splitting shows that the Cofactor centers have integer or zero electronic spin in the state M R. In order to characterize this state further we have studied the sample in strong applied fields. If S > 0 an applied field will polarize the electronic states giving rise to an internal magnetic field at the nucleus ~/int =--~-~ A/gnfJn, where (~) is an appropriately taken expectation value of the electronic spin (see for instance [14,15]). So for S > 0 the MSssbauer nucleus will sense an effective field J~e~ = ~int + ~ which is different from the applied field, while ~eff = / ~ for a diamagnetic compound. Fig. 5 shows a high-field MSssbauer spectrum of the cofactor centers in the state M a. It was prepared by taking a spectrum at 4.2 K in a parallel field of 55 kG and then subtracting the contributions of components D and Fe 2÷ (these components are diamagnetic and their spectra can be computed with adequate accuracy). The solid line in Fig. 5 is a theoretical spectrum of compoment M R assuming S = 0; the large magnetic splitting (Hin t ~ 100 kG) of the experimental spectrum clearly shows the presence of an internal magnetic field, i.e., S > 0 for the state M R.
132 l.-Z nW 0_ Z Z
o 1.o rn
-4
-2
0 2 VELOCITY ( m m / S )
4
Fig. 5. High-field s p e c t r u m o f t h e c o f a c t o r c e n t e r s in t h e s t a t e M R. T h e d a t a w e r e t a k e n at 4.2 K in a parallel field o f 50 k G . T h e c o n t r i b u t i o n s o f t h e d i a m a g n e t i c c o m p o n e n t s F e 2+ a n d D w e r e r e m o v e d . T h e s o l i d l l n e is a t h e o r e t i c a l s p e c t r u m a s s u m i n g d i a m a g n e t i s m (S = 0 ) for t h e s t a t e MR; it is a p p a r e n t t h a t t h e e l e c t r o n i c g r o u n d m a n i f o l d has S > 0.
Thionine oxidized MoFe protein The addition of thionine (6 e- equivalents per MoFe protein molecule) results in a one-electron oxidation of each cofactor center and each P-cluster. For azotobacter MoFe protein a thionine titration [6] proceeds in two distinct phases: In the first phase, four electrons are utilized to oxidize the four P-clusters from the state pN into the state P°X(E0' ~----250 mV). A second oxidation step, involving two electrons, transforms the cofactor centers into the state M °x. For clostridium MoFe protein the two processes are n o t as well separated presumably because the redox potential of the M°X/M N couple is substantially lower in clostridium MoFe protein [ 16] than in azotobacter MoFe protein. A series of MSssbauer spectra of thionine oxidized clostridium MoFe protein taken at 4.2 K are shown in Fig. 6. We have correlated [6] the components observed in Fig. 6 with those observed for the native enzyme. The central doublet in Fig. 6A results from the cofactor centers in the state M °x. The shape of the high-field spectra shows the absence of an internal magnetic field, i.e. the electronic ground state of M ° x is diamagnetic (S = 0). (We believe [6] that c o m p o n e n t S is also contained in the central doublet of Fig. 6A.) The remainder of the spectrum is a complex magnetic c o m p o n e n t (labeled M1). C o m p o n e n t M1 results from the P-clusters in the state pOX. We have discussed that M1 spectrum of azotobacter MoFe protein in considerable detail (see Appendix of Ref. 6) and we have given a spectral decomposition of the lowfield data (H < 1 kG). In the following we will extend the data analysis to clostridium MoFe protein and include an analysis of the high-field data. Before discussing the high-field spectra we briefly review some of the physics: In our interpretation a P-cluster in the state pOX contains 4 Fe atoms, spin-coupled to yield a system spin S. The spin S is as y e t undetermined, b u t is half integer and restricted to 3/2 ~< S ~< 9/2. The electronic ground manifold can be described by Eqn. 2 with D < 0 and k ~ 0. The lowest Kramers doublet is well separated (by at least 5 cm -1) from the first excited doublet; the ground doublet has a magnetic quantum number m = +S and effective g-values gx gy ~ 0 and gz = 4S (assuming go = 2 in Eqn. 2). A close inspection of the M1
133
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i
4
VELOCITY (mm/s) Fig. 6. L o w - t e m p e r a t u r e (4.2 K M ~ s s b a u e t s p e c t r a o f t h i o n i n e o x i d i z e d c l o s t r i d i u m M o F e p r o t e i n irL paraliel a p p l i e d fields. T h e c e n t r a l d o u b l e t in t h e 1 k G s p e c t r u m results f r o m t h e s t a t e M O X ; its a b s o r p t i o n p a t t e r n a t h i g h e r field i m p l i e s d i a m a g n e t i s m f o r t h e s t a t e M OX. T h e solid lines are t h e r e s u l t o f a s p e c t r a l d e c o m p o s i t i o n of t h e M1 s p e c t r u m using t h e p a r a m e t e r set in T a b l e I I I . We h a v e m a r k e d w i t h a r r o w s s o m e a b s o r p t i o n lines w h i c h b e l o n g t o c o m p o n e n t s 7 a n d 8 o f T a b l e I I I . T h e s e c o m p o n e n t s are c h a r a c t e r i z e d b y a p o s i t i v e m a g n e t i c h y p e r f i n e c o u p l i n g c o n s t a n t ; thei~ a b s o r p t i o n lines m o v e o u t w a r d ( H e f f i n c r e a s e s ) as t h e s t r e n g t h o f t h e a p p l i e d field increases.
spectrum reveals that it consists of a superposition of eight subspectra, suggesting two sets of slightly inequivalent P-clusters. Previously for azotobacter MoFe protein, we have restricted our data analysis to the low-field spectra, for three reasons: (1) Because k ~ 0 and D < 0 the expectation value of the elctronic spin for the ground doublet is non-zero only along the z
"gnflnHint Iz
(5)
with Hint = ""
134
to the zero-field splitting. Appreciable mixing would render the M1 spectrum unmanageably complex because it would produce finite values for (Sx) and (Sy). The direction of ~int relative to the electric field gradient tensor would then depend on the orientation of the molecule relative to the applied field. (If the magnetic hyperfine interaction in Eqn. 3 would be described by an A-tensor one would have to introduce further unknowns.) Level mixing would cause a broadening of the absorption lines of some subcomponents of M1 as the applied field is increased (the details depend on the anisotropy of the A-tensor and on the sign of its components). A close inspection (aided by computer simulations) of the spectra in Fig. 6 (and those taken on azotobacter MoFe protein) reveals that level mixing is negligible for applied fields up to 30 kG and of only minor importance at 55 kG. The observed broadening is almost entirely due to the nuclear Zeeman term in Eqn. 3. If this interpretation is correct one can compute the high-field spectra with the parameter set used to fit the lowfield spectra; the only additional unknowns are the signs of A0 for each subcomponent. We have pointed out earlier [6] that the M1 spectra of clostridium MoFe protein and azotobacter MoFe protein differ only in the MSssbauer parameters of one subcomponent. Thus a complete parameter set for clostridium MoFe protein was available from our azotobacter MoFe protein data analysis. A feature of the azotobacter MoFe protein decomposition was the assumption that half of the eight subcomponents were characterized by a positive hyperfine coupling constant A0. Although only two components with A0 > 0 were clearly evident in the 10 kG and 20 kG spectra, the preliminary analysis of the overall absorption pattern at higher fields suggested to us four components with
TABLE III HYPERFINE PARAMETERS SHOWN IN FIG. 6
AT 4.2 K USED
TO
GENERATE
THE
THEORETICAL
SPECTRA
F o r t h e s i m u l a t i o n s w e u s e d S = 5 / 2 , D = - - 1 0 c m - I , k = 0, a n d gO = 2 in E q n . 2. In t h e f r a m e w o r k of E q n s . 2 a n d 3 t h e i n t e r n a l m a g n e t i c field f o r H > 20 k G is given b y H i n t = +S Ao/gn{3 n, i.e. H i n t h a s the s a m e sign as A 0. T h e p a r a m e t e r a n d t h e E u l e r angles ~, ~ a n d 3, describing the o r e i n t a t i o n o f the local electric field gradient tensors relative to the e l e c t r o n i c s y s t e m are ~soft* p a r a m e t e r s (see A p p e n d i x of R e f . 6). We believe that the signs and m a g n i t u d e s o f H i n t are q u i t e reliable p a r a m e t e r s . A E Q a n d 5 f o r c o m p o n e n t s 7 a n d 8 are d e t e r m i n e d quite precisely f r o m zero-field spectra t a k e n a t 1 4 0 K ; to a c c o u n t f o r the s e c o n d order D o p p l e r shift 0 . 0 5 m m / s was a d d e d t o 5 t o o b t a i n t h e v a l u e s at 4 . 2 K. Q u a d r u p o l e splittings a n d i s o m e r i c shifts o f c o m p o n e n t s 1 - - 6 are less well d e t e r m i n e d . T h e m a g n i t u d e s o f H i n t a n d 5 f o r c o m p o n e n t 4 s e e m to us s o m e w h a t t o o small. T h e essential d i f f e r e n c e b e t w e e n the c l o s t r i d i u m M o F e protein a n d a z o t o b a c t e r M o F e p r o t e i n d a t a is t h e v a l u e of H i n t f o r c o m p o n e n t 2 (Hin t = - - 2 3 7 k G f o r a z o t o b a c t e r M o F e protein). Spectral component
Hint (kG)
AF.Q (mm/s)
6 (ram/s)
"Q
~
1 2 3 4 5 6 7 8
--287 --221 --245 --151 --259 --237 + 201 +223
--1.40 +1.53 +0.57 +0.60 + 1.26 --0.72 + 3.20 +2.30
0.56 0.64 0.40 0.25 0.48 0.49 0.65 0.68
0.70 0.05 0.42 0.32 0.70 0.70 0 0.66
90 0 0 0 0 90 0 0
,6
'T
--.90 18 62 4 58 --90 77 75
61 90 0 0 0 69 90 90
135
A0 > 0. After a systematic analysis of the azotobacter MoFe protein and clostridium MoFe protein high-field data we are now forced to the conclusion that only two subcomponents have A0 > 0. High-filed studies at T = 140 K have shown [7] that the iron sites which yield doublets I and II (the oxidized form of native component Fe 2÷) have A0 > 0. Thus the low-temperature components with A0 > 0 must have values for Z~EQand ~ which characterize doublets I and II. This assignment required a repartitioning of four subcomponents (3, 4, 7 and 8 of Table III in Ref. 6). We found a new spectral decomposition that fits the whole data set on clostridium MoFe protein and azotobacter MoFe protein remarkably well. In particular it properly describes the complex pattern of outward and inward moving absorption lines observed at higher fields. The solid lines in Fig. 6 are theoretical spectra computed from Eqns. 1--4 with the parameter set given in Table III. We have used S = 5/2 for the unknown electronic spin; as long as level mixing is negligible the particular value used for S is unimportant. We believe that our model of the P-clusters (D < 0, ~ ~ 0, four iron sites per P-cluster, two inequivalent sets in the state pO x) properly explains the relevant features of the observed spectra over a wide range of experimental conditions. It is virtually impossible to assign meaningful uncertainties to the parameters quoted in Table III. We have, however, tried to assess the situation by separat~ ing what we consider the more reliable parameters from the 'softer' ones. The softness of parameters is primarily related to the fundamental ambiguity problem discussed in Ref. 6. The parameter set quoted in Table III should be a useful reference for researchers studying nitrogenase from other organisms. Discussion The MSssbauer data obtained for azotobacterMoFe protein and clostridium MoFe protein are strikingly similar, suggesting practically identical metal centers in both proteins *. Our studies have led to a remarkably consistent picture; the holoenzymes (Mr 220 000--250 000) contains two M-centers and four P-clusters accounting for 28 of 30 Fe atoms. The cofactor centers M are novel structures which have been stabilized in three oxidation states. The state MN, observed for the protein as isolated, has electronic spin S = 3/2. All properties connected with an S = 3/2 spin system have been observed with both EPR and MSssbauer spectroscopy: The prominent EPR signals around g = 4.3, 3.7, and 2.0 and the MSssbauer spectra of Fig. 3 result from the m = +1/2 ground doublet. An EPR signal at g = 5.85 [11] and some MSssbauer absorption lines (for clostridium MoFe protein, Huynh, B.H. and Miinck, E., unpublished data) have also been observed for the
* This s t a t e m e n t refers t o t h e structural p r o p e r t i e s o f t h e c e n t e r s as e l i c i t e d b y E P R a n d M ~ s s b a u e r s p e c t r o s c o p y . O n o t h e r levels p r o n o u n c e d d i f f e r e n c e s b e t w e e n t h e p r o t e i n s have b e e n r e p o r t e d . F o r i n s t a n c e , t h e P o t e n t i a l o f t h e M O X / M N c o u p l e is 1 4 0 m V l o w e r in klebsiella Mol~e p r o t e i n t h a n in a z o t o b a c t e r M o F e p r o t e i n [ 1 6 ] . A l s o c r o s s - r e a c t i o n s b e t w e e n F e p r o t e i n s and M o F e p r o t e i n s [ 1 7 ] r e v e a l p a t t e r n s w h i c h s e e m t o r e f l e c t t h e d o m i n a t i n g i n f l u e n c e o f t h e p r o t e i n s t r u c t u r e rather t h a n s t r u c t u r a l features of the MoFe protein metal centers.
136 m = +3/2 excited state doublet. Both techniques have yielded, within the uncertainties, the same value for the zero-field splitting parameter D = +(6 + 1.5) cm -1 for both azotobacter MoFe protein and clostridium MoFe protein. The spin S = 3/2, which refers to the cluster as a whole, results from spincoupling of (most likely) six Fe atoms and (presumably) one Mo atom. Spincoupling is proven by the fact that the spin is shared by six iron atoms and by the observation of positive and negative hyperfine coupling constants. (Positive coupling constants result when the individual spin is oriented antiparallel to the system spin.) Although all iron atoms o f the M-centers have nearly the same quadrupole splitting AEq they are quite inequivalent when their magnetic properties are examined; the magnitudes of Ao/gn~n range from 75 kG to 145 kG. Extended X-ray absorption fine structure data [18] implicate m o l y b d e n u m as a structural c o m p o n e n t of the M-centers. Experiments with samples enriched in 9SMo have failed to show any broadening of the EPR lines due to hyperfine interactions of the 9SMo nucleus with the electronic spin of the M-center. These results are somewhat disturbing; however, they constitute no evidence against the presence of m o l y b d e n u m in the M-centers, since peculiarities of the spincoupling mechanism or 9SMo quadrupolar effects could be at the r o o t of this problem. One-electron redox reactions yield the states M °x (S = 0) and M a (S > 0, integer). The quadrupole doublets observed in the states M a and M °x are n o t as sharp as that observed in the state M N, implying that the individual subsites are somewhat inequivalent (see Table I). The (average) isomeric shift increases by a b o u t 0.06 mm/s per electron added to the cluster. For comparison, the average isomeric shift of 4Fe-4S centers changes by a b o u t 0.15 mm/s per one-electron redox step [19]. In the native enzyme and under fixing conditions the P-clusters are observed in the state pN. A P-cluster in this oxidation state contains three irons of the D-type and one iron site yielding c o m p o n e n t Fe :÷. W e have studied both MSssbauer components in azotobacter MoFe protein and clostridium MoFe protein in applied fields up to 55 kG in the temperature range from 1.5 to 200 K. These studies firmly establish the diamagnetism of the state pN. To within +0.02 mm/s all 12 D-irons of azotobacter MoFe protein exhibit a quadrupole splitting AEQ = 0.81 mm/s. Curiously, all D-irons of clostridium MoFe protein have, within the same narrow margins, AEQ = 0.70 mm/s. Thus the environments of t h e P-clusters differ in both proteins such that all D-irons are affected in the same way. A one-electron oxidation transforms the P-clusters into the state pOX which has half-integer electronic spin 3/2 ~< S ~< 9/2. It is n o t e w o r t h y that the state pOX is EPR-sflent for all proteins investigated so far. We have suggested [6] that a large and negative zero-field splitting parameter D, in conjunction with ), ~ 0, together with fast relaxation for T > 10 K is responsible for this observation. This suggestion is supported by the present study: The analysis of the high-field data suggests a large value for D. For instance, for S = 5/2 our analysis yields [DI> 5 cm-*. For D = - - 5 cm -1 the EPR-active state (which would yield resonances around g = 6 and g = 2) would be 6D -- 30 cm -1 ~ 42 K above the ground state and therefore only marginally populated at T < 10 K. The
137
electronic spin S is as yet undetermined *. Using the information obtained from the M5ssbauer studies the magnetic susceptibility, for T <: 6 K, is predicted to be entirely due to the lowest Kramers doublet and is given by X = S2{J2/kT per P-cluster (assuming go = 2 in Eqn. 2 and H < < kT). Thus lowtemperature susceptibility studies on thionine oxidized MoFe protein (the state M°x is diamagnetic) could be used to determined the unknown spin. The high-field MSssbauer studies and the quantitation of the azotobacter MoFe protein thionine titrations [6] prove independently that the P-clusters contain spin-coupled iron atoms. The analysis of the high-field MSssbauer data shows that one iron site of a P-cluster (the site yielding M5ssbauer component Fe 2÷) has a positive A0 while the other three are characterized by A0 < 0. In contrast, the oxidized high-potential-iron-protein from Chromatium vinosum and the reduced 4Fe-4S Bacillus stearothermophilus ferredoxin apparently have two iron atoms each with positive and negative A0 [19]. Component S is present in equal amounts (about 6% of the total Fe, or 2Fe atoms) in azotobacter MoFe protein, clostridium MoFe protein, and apparently also in klebsiella MoFe protein with practically identical M5ssbauer parameters (AEQ ~-- 1.35 mm/s and 8 = 0.60 ram/s). The MSssbauer parameters are quite atypical of commonly observed impurities (high-spin ferric or ferrous ions). Also no EPR signal attributable to S has been observed despite the fact that samples have been studied over a wide range of redox conditions. The iron atoms of component S are certainly not a structural part of the cofactor centers. This is proven by the fact that the component S (or a component with similar isomeric shift) is absent in isolated cofactor material [11]. It is also quite unlikely, although not strictly ruled out, that S is a structural component of the P-clusters; in that case, however, the M1 spectrum would have to account for 60% of the total absorption, in contrast to the experimental results (52--55%). A further elucidation of the nature of component S will be achieved when reconstituted MoFe protein preparations with selective STFe enrichment for the M- or P-clusters become available. Acknowledgements This work was supported by the National Science Foundation grant PCM08522, by the National Institutes of Health through grant GM17170, by the Graduate Research Committee of the University of Wisconsin, and by Research Career Development Award K04-GM70683 (E.M.). W.H.O-J. is an I.H. Romnes Faculty Fellow of the University of Wisconsin. References 1 O n - h e - J o h n s o n , W . H . , H a m i l t o n , W . D . , L j o n e s , T., Tso, M.-Y.W., B u r r i s , R.H.~ S h a h , V . K . a n d Brill, W . J . ( 1 9 7 2 ) P r o c . N a t l . A c a d . Sci. U . S . A . 6 9 , 3 1 4 2 - - 3 1 4 5 * We ha~e o b s e r v e d r e p r o d u c i b l y a w e a k , t r a n s i e n t E P R signal w i t h g-values a r o u n d 6 . 3 a n d 5 . 6 w h i c h results f r o m a n S = 5 / 2 s y s t e m w i t h k ~ 0 . 0 2 . T h e t e m p e r a t u r e d e p e n d e n c e o f the signal establishes f i r m l y t h a t it r e s u l t s f r o m an e x c i t e d electronic state (thus e x c l u d i n g a h e m e i m p u r i t y ) , s e p a r a t e d i n energy b y a b o u t 3 0 c m - I f r o m t h e g r o u n d s t a t e . T h u s t h e s i g n a l r e s u l t s f r o m a n e l e c t r o n i c s y s t e m w i t h the s a m e properties as that o f t h e state p O X ( E m p t a g e , M., O r m e * J o h n s o n , W . H . , H u y n h , B . H . , a n d M i l n c k , E., u n p u b l i s h e d r e s u l t s ) .
138 2 Zumft, W.G. and Mortenson, L.E. (1975) Biochim. Biophys. Acta 416, 1--55 3 Winter, H.C. and Burrls, R.H. (1976) Annu. Rev. Biochem. 45, 409--426 4 0 r m e - J o h n s o n , W.H., Davis, L.C., Henzl, M.T., Avernl, B.A., Orme-Johnson, N.R., Miinck, E. and Zimmermann, R. (1977) in Recent Developments in Nitrogen Fixation (Newton, W., Postgate, J.R. and Rodriguez-Barrueco, C., eds.), pp. 131--178, Academic Press, New York 5 Milnck, E., Rhodes, H., Orme-Johnson, W.H., Davis, L.C., Brill, W.J. and Shah, V.K. (1975) Biochim. Biophys. Acta 400, 32--53 6 Zimmernann, R., Miinck, E., Brill, W.J., Shah, V.K., Henzl, M.T., Rawlings, J. and Orme-Johnson, W.H. (1978) Biochim. Biophys. Acta 537, 185--207 7 Huynh, B.H, M(inck, E. and Orme-Johnson, W.H. (1979) Biochirn. Biophys. Acta 527, 192--203 8 Zumft, W.G., Mortenson, L.E. and Palmer, G. (1974) Eur, J. Biochem. 46, 525--535 9 Donald, C., Passey, B.I. and Swaby, R.J. (1952) J. Gen. Microbiol. 7, 211--220 10 Emptage, M.H., Zimmermann, R., Que, L., Mflnck, E., Hamilton, W.D. and Orme-Johnson, W.H. (1977) Biochim. Biophys. Acta 495, 12--23 11 Rawlings, J., Shah, V.K., Chisneil, J.R., BI"JlI, W.J., Zimmermann, R., Milnck, E. and Orme-Johnson, W.H. (1978) J. Biol. Chem. 253, 1001--1004 12 Orme-Johnson, W.H. and Davis, L.C. (1977) in Iron-Sulfur Proteins (Lovenberg, W., ed.), Vol. 3, pp. 15--60, Academic Press, New York 13 Smith, B.E. and Lang, G. (1974) Biochem. J. 137, 169--180 14 Milnck, E. (1978) in The Porphyrins (Dolphin, D., ed.), Vol. IV, Chapter 8, pp. 379---423, Academic Press, New York 15 Lang, G. (1970) Q. Rev. Biophys. 3, 1--60 16 O'Donnell, M.J. and Smith, B.E. (1978) Biochem. J. 137, 831--839 17 Emmerich, D.W. and Burris, R.H. (1978) J. Bacteriol. 134,936--943 18 Cramer, S.P., Hodgson, K.O., GUlum, W.O. and Mortenson, L.E. (1978) J. Am. Chem. Soc. 100, 3398--3407 19 C a m m a c k , R., Dickson, D.P.E. and Johnson, C.E. (1977) in Iron-Sulfur Proteins (Lovenberg, W., ed.), Irol. 3, pp. 283--330, Academic Press, New York