Mössbauer studies of cytochrome c-551

502

Biochimica et Biophysica Acta~ 576 (1979) 502--508 © Elsevier/North-Holland Biomedical Press

BBA Report BBA 31269

MOSSBAUER STUDIES OF CYTOCHROME c-551 INTRINSIC H E T E R O G E N E I T Y R E L A T E D TO g-STRAIN

A. DWIVEDI, W.A. TOSCANO, Jr. and P.G. DEBRUNNER

Departments of Physics and Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801 (U.S.A.) (Received September 12th, 1978)

Key words: Cytochrome c-551; M6ssbauer analysis; Low spin heme

Summary The MSssbauer spectra of oxidized and reduced cytochrome c-551 from Pseudomonas aeruginosa are analyzed. Excess broadening is observed in the 4.2 K spectra of oxidized c-551 which is consistent with a Gaussian distribution of the crystal field parameters A and R as inferred from the g-strain model of EPR line shapes.

Recent progress [1--5] in the characterization of c-type cytochromes [6] has stimulated interest in the mechanism of action of these proteins [7] and its relation to the properties of the active site [3]. The three-dimensional structures of several proteins of this class have been determined [ 1 ], and the electronic properties of the active centers have been studied b y various techniques [2--5,8,9]. Below we present MSssbauer data on a bacterial c-type c y t o c h r o m e and we show that a satisfactory parametrization of the spectra is obtained if the g-strain inferred from an analysis of the EPR line shapes [11 ] is taken into account. In earlier MSssbauer studies of c y t o c h r o m e c from Torula utilis [8] and cz from Rhodospirillum rubrum [5] discrepancies were noted between the experimental spectra and the simulations based on model calculations. The same deficiencies of the model are found in the case of c-551. Huynh et al. [5] correctly associated these discrepancies with an intrinsic inhomogeneity of the protein sample. Our approach follows their suggestion and demonstrates that the heterogeneity can be adequately described by a Gaussian distribution of the rhombic (and tetragonal) crystal field splittings, which in turn can be derived from the shape of the EPR spectrum.

503 Thus a considerable improvement of the MSssbauer simulations is obtained which allows a more reliable determination of the hyperfine parameters. We have studied cytochrome c-551 from Pseudomonas aeruginosa (M = 8900) which contains a single heme group and functions as an electron donor in the oxidase/nitrite reductase system [6,10]. Cytochrome c-551 has the same overall folding pattern, apart from the deletion of one loop, as the eukaryotic c-cytochromes [ 1 ]. Moreover, the local environment of the active site is quite similar in c-551, cytochrome c2 from R. rubrum and the c-cytochromes from eukaryotes. Specifically, the heme group is bound to the polypeptide chain through two cysteine linkages, and the heme iron has histidine and a methionine as axial ligands [ 1 ]. As in other c-type cytochromes, the iron is in the low-spin state both in the oxidized (Fe 3+, S = ~) and the reduced (Fe 2+, S -- 0) protein. The two g-values reported for oxidized c-551, gl = 3.2 and g2 = 2.05 at pH 4.9 [11] match those of bakers' yeast iso-2, Euglena, c2 of R. rubrum and c-550 of Paracoccus denitrificans, gl ~ 3.2, g2 = 2.05, g3 ~ 1.39 [9], and we expect the unobserved third g-value of c-551 to be close to 1.3. Cultures of P. aeruginosa were grown anaerobically on an 57Fe-enriched medium, and c y t o c h r o m e c-551 was isolated and purified by methods adapted from published procedures [10]. The samples were frozen solutions of 1.9 mM c-551 in acetate buffer, pH 4.9. Reduced c-551 was prepared by addition of a small a m o u n t of dithionite to the native protein. MSssbauer spectra were recorded on a constant acceleration spectrometer with the sample mounted in a Janis variable-temperature cryostat or an immersion cryostat with a superconducting magnet. Doppler shifts were calibrated periodically with an iron foil, and all velocities are given relative to the center of gravity of metallic iron at 300 K. The low-temperature spectra of oxidized c-551 (Fig. 1) show resolved magnetic hyperfine splitting typical of low-spin ferric iron, S = 1/2, while at 180 K (Fig. 2a), the magnetic interaction averages out, leaving a single, somewhat broadened quadrupole doublet. Table I lists the quadrupole splitting AEQ, isomer shift ~ and linewidth F derived by least-squares fitting two Lorentzians to the 180 K data. Fig. 2 shows the spectra of reduced c-551 at 4.2 K in a very weak field (2b) and in a strong field (2c). Solid lines are simulations based on the parameters listed in Table I assuming a diamagnetic complex. The isomer shift and the quadrupole tensor are quite similar to those of other c-type cytochromes [5,8]. The complex, low-temperature spectra of oxidized c-551, Fig. 1, contain considerably more information than those of Fig. 2, and their analysis involves a number of approximations. The complexity of the spectra arises from the simultaneous presence of magnetic and electric hyperfine interaction, described by the Hamiltonian ~=

~ ~'g'~

+ ~'+~'f'+ l"P'f-~n

gn ~ - T

(1)

Here ~" is the effective spin operator of the low-spin ferric iron, S = 1/2, ~+the

504 I

I

I

I

i

i

0.0

0, I

0,2

(o) 0,3

LI.J

O.q

Q= aZ

0.0

", h L~

0,1

v

!l

I

0.2 (b)

0.3

O.LI

0.5

0.6 I

i

I

I

I

-IO

-S

0

S

I0

VELOCITY

IN

(MM/SEC)

Fig. I . M ~ s s b a u e r s p e c t r a o f f e r r i c y t o c h r o m e c - 5 5 1 at 4 . 2 K in an e x t e r n a l field o f 4 4 0 G applied (a) parallel and (b) p e r p e n d i c u l a r to t h e d i r e c t i o n o f the g a m m a rays. T h e solid lines are s i m u l a t i o n s b a s e d o n a d i s t r i b u t i o n o f p a r a m e t e r s c e n t e r e d o n t h e values given in T a b l e I, w h i l e t h e d o t t e d lines are b a s e d o n the d i s c r e t e values listed. D e t a i l s are given in t h e t e x t .

correspo_.nding g-tensor, • the nuclear spin operator, ~ the magnetic hyperfine tensor, I . ~ 5 . I ' represents the quadrupole interaction, also written as I.P.I

,= ( e Q / 6 ) ( V x x I x =

(eQVzz/12)[3Iz

2 + Y y y I y 2 + V z z I z 2) = 2 -

15/4 +

~7(Ix 2 -

iy2)]

(2)

with ~ = ( V x x - V y y ) / V z z , and the last term is the nuclear Zeeman interaction. Ti~e weak magnetic field, H = 440 G, applied in the measurements of Fig. 1

505 -

I

I

I

I

I

I

0.0

0.5

1.0

1.5

bJ u

2.0

hl Q_ Z

LU b_ b_ I,I

0.0

0.5

(b)

0.0

0.5

1.0 (c) 1.5

2.0

2.5

3.0

I

I

I

I

-3.0

-~.0

-I.0

0.0

1.0

I

I

2.0

3.0

VELOCITY IN {MM/SEC} Fig. 2. M~Jssbauer s p e c t r a o f o x i d i z e d c y t o c h r o m e c - 5 5 1 a t 1 8 0 K (a) a n d o f r e d u c e d c y t o c h r o m e ¢ - 5 5 1 in a field o f 4 4 0 G (b) a n d 2 3 . 6 k G p a r a l l e l t o t h e g a m m a r a y s (c). Solid Lines a r e s i m u l a t i o n s b a s e d o n t h e p a r a m e t e r s listed i n T a b l e I.

506 TABLE I MOSSBAUER PARAMETERS OF CYTOCHROME c-551 AND RELATED QUANTITIES N u m b e r s in parenthesis are uncertainties in units of the least significant digit.

6 a (ram/s) ~EQ (ram/s) FWHM(mm/s)

Ferrocytochrome

Ferrlcytochrome

4.2 K

200 K

css 1

c2 b

0.45(5) 1.29(5) 0.5(2) 0.26(1)

0.41(2) 1.33(5)

0.23(2) c 2.03 (2) c -0.9 0.35(1) c

0.25 2.26 -1.5

(1.25 d, 2.05, 3.2) (--32, 29, 66) 3.59 1.29

(1.23, 2.11, 3.13) (--35, 25, 62.5) 3.08 1.33

~_,

A/gn~ n) (T) R

aRelative to metallic iron at 300 K. bvalues from H u y n h et al. [5] for c y t o c h r o m e c 2 from Rhodospirillurn rubrurn. CMeasured at 180 K. dcalculated from gy and gz.

effectively decouples the electronic and nuclear spins. As a result, the Hamiltonian q~ of Eqn. 1 separates into an electronic and a nuclear part,

~ = ~e +~n,

~e =/3 ~'g'/~,

~n = <~./~.~*+ ~.jS".~

(3)

where <~> is now the spin expectation value calculated for the two eigenstates Of~e, and the last term of Eqn. 1 has been dropped since it is negligible compared with the others. Given the tensors ~, X and Vii theoretical MSssbauer spectra can be calculated using standard programs [12]. We next use the Griffith model [13] to derive the third g value of c-551 and Lang's extension of this model [14,15] to estimate ~ and Vii. The Griffith model describes the ground state of the low-spin ferric iron in terms of only two parameters, a tetragonal and a rhombic crystal-field c o m p o n e n t ;~A and kR, respectively, measured in units of the spin orbit coupling constant ),. Both components are assumed to be coaxial with the dominant octahedral field and comparable in magnitude with the spin-orbit coupling k~'s*. The result is a set of three Kramers doublets which are linear combinations of the t2g orbitals Ixy>, Ixz> and lyz> with coefficients completely determined by ~. The two known g-values of c-551, together with the normalization condition, suffice to fix the three coefficients of the wave function. With the assignment gz = 3.2, gy = 2.05 [16], the model implies the crystal field parameters ~.0 = 3.59, R0 = 1.29, and it predicts gx = 1.25 and the hyperfine tensors (V)/(gn{Jn) = (-42.1, 18.6, 76.7) T, AEQ = 3.18 mm/s, ~ = -3.09. Here we have used the parameters 2(~ /~0/4~ = 71 T, ~ = 0.35, (2/7) e 2 Q ( 1 - R ) ( c / E ~ ) = 4.75 mm/s, (k Q(1-~( )/)(c/E~)~ = 0.0735 mm/s [ 1 4 , 1 5 ] To f u r t h e r improve the simulations we treated A and Vii a s adjustable p**arameters and optimized them by trial and error to obtain the final values = t~ (v) + A ~ , Vii = Vii (G) + A Vii listed in Table I. (Vii is specified implici~y in terms of ~ and AEQ = Vzz(1 + 1/3~2)'~.) As before the principal axes of g, and Vii were assumed to coincide. The best simulations, shown as dotted ]jr.es Jn Fig. 1, match the data quite weii except for the outer peaks which

507 have the wrong shape, too large an amplitude and too small a width. An overall increase of linewidth does not improve the fit. Similar problems were encountered in the analysis of c y t o c h r o m e c2 by Huynh et al. [ 5]. These authors suggested that the excess broadening of the outer lines is due to an intrinsic heterogeneity of the protein, in other words, they propose that each molecule may have a different g-tensor and hyperfine interaction. If this conjecture is correct, a quantitative analysis of the spectra requires some assumptions about the distribution of g, .~ and Vii, i.e. additional parameters have to be introduced. This task is most easily accomplished by resorting to the Griffith model, assuming that the sample heterogeneity is reflected in a normal distribution of the crystal-field splittings A and R. This approach is strongly suggested by the success of the analogous g-strain model of EPR line shape [ 11 ]. According to this model the dominant contribution to the linewidth in the low-temperature EPR spectra of spin S = 1/2 heme proteins arises from g-strain, i.e. from a distribution of g-values. The latter can be parametrized by a Gaussian distribution of crystal-field splittings about their mean 40 and R0 with rms deviations a~ and aR. For c y t o c h r o m e c in particular, this analysis works well with a~ = 0.167, OR = 0.207 [11]. We use these values since no definite EPR spectra are available for c-551 yet. Adopting this g-strain model we arrived at the final simulations shown as solid lines in Fig. 1. Specifically, the quantities g(A0, R), ~(G)(A0, R) and Vii(V)(Ao, R) were calculated from the Griffith model [ 1 3 - - 1 5 ] f o r nine different values of R in the range Ro + 2OR. Variations of A affect g, ~ and Vii very little and were therefore ignored. MSssbauer spectra calculated for the corresponding values g(~0, R), .~(V)(~0, R) + ~.~ and Vii(G)(Ao, R) + A Vi i were added with appropriate weighting factors. Clearly the agreement with the data is quite good leaving no doubt that the same sample heterogeneity that causes the g-strain width of the EPR lines is also responsible for the excess broadening observed in the MSssbauer spectra. While the most reasonable way to model the parameter distribution needs further study, the present approach appears to be adequate for c-551. It definitely improves the simulation and permits a more precise determination of hyperfine parameters, thus opening the way to a systematic comparison of the active site structure in different cytochromes. The intriguing question concerning the origin of the heterogeneity in crystal-field splitting can only briefly be mentioned. Is it a freezing artifact that reflects the response of the protein to some external stress, or is it a frozen-in distribution of conformational states, or both? In either case the intrinsic heterogeneity is a potentially important property of the system, which may have some bearing on other experiments [17] and certainly calls for further investigation. This research was supported by grants from the U.S. Public Health Service, USPH GM 16406, and the National Science Foundation, NSF PCM 76-81025. References 1 Almassy, R.J. and Dickerson, R.E. (1978) Proc. Natl. Acad. Sci. U.S. 75, 2674---2678 2 Keller, R.M. and W/ithrich, K. (1970) Biochim. Biophys. Acta 533, 195--208

508 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17

Keller, R.M. and W~ithrich, K. (1978) Biochem. Biophys. Res. Commun. 83, 1132--1139 Scholes, C.P. and Van Camp, H.L. (1976) Biochim. Biophys. Acta 434, 290---296 Huynh, B.H., Emptage, M.H. and Milnck, E. (1978) Biochim. Biophys. Acta 534, 295--306 Dickerson, R.E° and Timkovich, R. (1975) in The Enzymes (Boyer, P., ed.), 397--547, Academic Press, New York Pettigrew, G. (1978) FEBS Lett. 86, 14--16 Lang, G., Herbert, D. and Yonetani, T. (1968) J. Chem. Phys. 49, 944---950 Brautigan, D.L., Feinberg, B.A., Hoffrnan, B.M., Margoliash, E., Peisach, J. and Blumberg, W.E. (1977) J. Biol. Chem. 252, 574--582 , Toscano, W.A., Jr. (1977) Ph.D. Thesis, University of Illinois Schweitzer, K., Devaney, P., Wagner, G. and Debrunner, P.G. (1979) J. Magn. Res., submitted Miinck, E., Groves, J.L., Tumolino, T.A. and D e b ~ n n e r , P.G. (1973) Computer Phys. Commun. 5, 225--238 Grlffith, J.S. (1957) Nature 180, 30--31 Lap.g, G. and Marshall, W. (1966) Proc. Phys. Soc. 87, 3--34 Oosterhuis, W.T. and Lang, G. (1969) Phys. Rev. 178, 439--456 Taylor, C.P.S. (1977) Biochim. Biophys. Acta 491,137--149 Austin, R.H., Bceson, K., Eisenstein, L., Frauenfelder, H., Gunsalus, I.C. and Marshall, V.P. (1973) Phys. Rev. Lett. 32, 403--405