[14] Rubredoxin in crystalline state

[14]

RUBREDOXIN IN CRYSTALLINE STATE

203

and D. vulgaris confirmed this idea. 56Both apoflavodoxins bind 3',5'-FBP somewhat more weakly than 5'-FMN owing to slower association rate constants. The redox potentials of the artificial complex, however, are similar to the values reported for the native protein. 31p NMR experiments revealed that the 3'-phosphate group is accessible for Mn 2÷ and indicates that this group is located close to the protein surface. 56 This is in sharp contrast with the 5'-phosphate group, which is dianionic 6° and buried in both proteins. The 5'-phosphate group therefore determines the specificity of flavin binding, whereas introduction of the 3'-phosphate group only slightly influences the protein conformation. The latter conclusion is in full accordance with results obtained from the crystal structure of D. vulgaris flavodoxin as well as ~H NMR results, which show that the 3'-OH of the ribityl side chain is oriented toward bulk solvent. 48,64 64 W. Watt, A. Tulinsky, R. P. Swenson, and K. D. Wautenpaugh, J. Mol. Biol. 218, 195 (1991).

[14] R u b r e d o x i n in C r y s t a l l i n e S t a t e By

LARRY C . SIEKER, RONALD

E.

STENKAMP,

and

JEAN L E G A L L

General Remarks Rubredoxin (Rd) is one of the simplest of iron proteins and has been found, thus far, only in certain microorganisms. 1 The initial report and characterization of a rubredoxin was done by Lovenberg and Sobel in 1965. 2 Rubredoxins are composed of 45 to 54 amino acid residues with molecular weights ranging from 5000 to 6000 and contain 1 iron atom liganded by 4 cysteine residues. The iron center can be reversibly reduced at a redox potential near 0 inV.1 Although many rubredoxins have been detected and isolated from a variety of bacteria, only 13 of the rubredoxins have had amino acid sequences determined. Figure 1 shows the amino acid sequence alignment of the 13 rubredoxins. Because this chapter is primarily directed to the sulfate-reducing bacteria we have chosen to divide these rubredoxins into three categories. Figure la lists the Rds from the sulfate-reducing Desulfooibrio species, Fig. lb shows the Rds from a mixed assortment of bacteria, and Fig. lc contains the thermophilic Rds. i T. G . Spiro, "Iron-Sulfur Proteins." Wiley (Interscience), New York, 1982. 2 W. Lovenberg and B. E. Sobel, Proc. Natl. Acad. Sci. U.S.A. 54, 193 (1965).

METHODS IN ENZYMOLOGY,VOL. 243

Copyright© 1994by AcademicPress, Inc. All rightsof reproductionin any formreserved.

204

1

D.vH

*

RUBREDOXIN

SEQUI~CE

20

30

10

COMPARISON

40

50

+ +++ + + + ++ ++ + MKKYVCTVCGYEYDPAEGDPDNGVKPGTSFDDLPADWVCPVCGAPKSEFEAA

I

Ill lltlIlll:

I

I

l I II II

fMKKYVCTVCGYEYDPAEGDPDNGVKPGTAFEDVPADWVCPICGAPKSEFEPA

D.gs

* fMDIYVCTVCGYEYDPAKGDPDSGIKPGTKFEDLPDDWACPVCGASKDAFEKQ

I I

rll Illlllll: III IIIlllll:

* fMQKYVCNVCGYEYDPAEHD

1

C.pa

i0

I

I

I

I

I I

I II

II

I

I IJ II

I

l

I

....... NVPFDQLPDDWCCPVCGVSKDQFSPA

20

30

40

50

II

l

I

II

il

t

i

MKKFICDVCGYIYDPAVGDPDNGVEPGTEFKDIPDDWVCPLCGVDKSQFSETEE

II P.as

II

I

I

It

II

I

I

MQKFECTLCGYIYDPALVGPDTPDQDG-AFEDVSENWVCPLCGAGKEDFEVYED

II

II

I

I

II

II

I

Ch.t

MQKYVCSVCGYVYDPADGEPDDPIDPGTGFEDLPDEWVCPVCGVDKDLFEPES

B.me

MQKYVCDICGYVYDPAVGDPDNGVAPGTAFADLPEDWVCPECGVSKDEFSPEA

II

II

If M. e l

II

I

I

I

I

M D K Y E C S I C G Y I Y D E A E G D -D G N V A A G T K F A D L

If C.ts

II

I

t

II

II

II

II

I I

I I I

PADWVCPTCGADKDAFVKMD

II

II

I

I

fMTKYVCTVCGYVYDPEVGDPDNNINPGTSFQDIPEDWVCPLCGVGKDQFEEEA

1

*

l0

20

30

40

50

+ +++ + + + ++ ++ + + -AKWVCKICGYIYDEDAGDPDNGISPGTKFEELPDDWVCPICGAPKSEFEKLED

lJ I C. t h

I

+ +++ + + + ++ ++ + + * fMKKYTCTVCGYIYNPEDGDPDNGVNPGTDFKDIPDDWVCPLCGVGKDQFEEVEE

C.pf

P. f u

+

I

D.vM

D.ds

[14]

DISSIMILATORY SULFATE REDUCTION

Irilrl

ilJ

fMEKWQCTVCGYIYDPEVGDPTQNI

I flJlll Jlllflll lJ

I

III

PPGTKFEDLPDDWVCPDCGVGKDQFEKI

FIG. 1. *, Crystal structures;l, conserved residues among the various types ofrubredoxins; +, residues that are strictly conserved throughout the 13 rubredoxins. (a) Desulfovibrio: D.vH, Desulfovibrio vulgaris Hildenborough [M. Bruschi, Biochem. Biophys. Acta 434, 4 (1976); G. Voordouw, Gene 67, 75 (1988)]; D.vM, Desulfovibrio vulgaris Miyazaki IF. Shimizu, M. Ogata, T. Yagi, S. Wakabayashi, and H. Matsubara, Biochemie 71, 1171 (1989)]; D.gs, Desulfovibrio gigas [M. Bruschi, Biochem. Biophys. Res. Commun. 70, 615 (1976)]; D.ds, Desulfovibrio desulfuricans IS. Hormel, K. A. Walsh, B. C. Prickril, K. Titani, and J. Le Gall, FEBS Lett. 201, 147 (1986)]. (b) Mixed bacteria: C.pa, Clostridiumpasteurianum [K. T. Yasunobu and M. Tanaka, in '+Iron-Sulfur Proteins" (W. Lovenberg, ed.), p. 27. Academic Press, New York, 1973; I. Mathieu, J. Meyer, and J.-M. Moulis, Biochem. J. 285,255 (1992)]; C.pf, Clostridium perfringens [Y. Seki, S. Seki, M. Satoh, A. Ikeda, and M. Ishimoto, J. Biochem. (Tokyo) 106, 336 (1989)]; P.as, Peptostreptococcus asaccharolyticus (formerly Peptococcus aerogenes) [H. Bachmeyer, A. M. Bensen, K. T. Yasunobu, W. T. Garrard, and H. R. Whitely, Biochemistry 7, 986 (1967)]; Ch.t, Chlorobium thiosulfatophilum [K. J. Woolley and T. E. Meyer, Eur. J. Biochem. 163, 161 (1987)]; B.me, Butyribacterium methylotrophicum [K. Saeki, Y. Yao, S. Wakabayashi, G. J. Shen, J. G. Zeikus, and H. Matsubara, J. Biochem. (Tokyo) 106, 656 (1989)]; M.el, Megasphaera elsdenii [H. Bachmeyer, K. T. Yasunobu, J. L. Peel, and S. Mayhew, J. Biol. Chem. 243, 1022 (1968)]; C.st, Clostridium sticklandii [sequence reported by I. Mathieu, J. Meyer, and J.-M. Moulis, Biochem. J. 285, 285 (1992)]. (c) Thermophiles: P.fu, Pyrococcus furiosus [P. R. Blake,

[14]

RUBREDOXIN IN CRYSTALLINE STATE

205

Physiological Role The redox potentials of Rds isolated from sulfate-reducing bacteria are relatively high ( - 5 to 0 mV) 3 whereas dissimulatory sulfate reduction requires electrons from -400 to -200 m V . 4'5 Consequently, the search for the electron transfer reaction(s) catalyzed by Rds is a difficult task. Rubredoxins have been proposed to accept electrons from carbon monoxide dehydrogenase (EC 1.2.99.2) in other anaerobes such as Clostridium thermoaceticum or Acetobacter woodii. 6For the sulfate-reducing bacteria, such an activity had not been detected, nor had electron donors been clearly linked to sulfate respiration. It was shown several years ago that the Rd from Desulfovibrio gigas is reduced by the tetraheme cytochrome c3 from the same organism 7 in the presence of hydrogenase. A hypothetical model of the complex formed between these two proteins from Desulfovibrio vulgaris Hildenborough has been proposed utilizing computer graphic modeling and nuclear magnetic resonance (NMR) spectroscopy. 8 According to this model, the iron atom of Rd is in close proximity to heme 1 (the most positive heme) of the cytochrome. More importantly, D. gigas cells that have been grown on a lactate-sulfate medium contain an NADHrubredoxin oxidoreductase (NRO). 9 This enzyme is composed of two subunits of 27 and 32 kDa, respectively 1° and contains both FAD and riboflavin 5'-phosphate (FMN). It induces the specific reduction of D. gigas Rd. Rubredoxins from other Desulfooibrio species show low reaction rates with this enzyme. Such a specificity can be explained by the differences that exist between Rds in terms of the external residues. 3 I. Moura, J. J. G. Moura, H. M. Santos, A. V. Xavier, and J. Le Gall, FEBS Lett. 107, 419 (1979). 4 j. Le Gall, J. J. G. Moura, H. D. Peck, Jr., and A. V. Xavier, in "Iron-Sulfur Proteins" (T. G. Spiro, ed.), p. 177. Wiley, New York, 1982. 5 j. Le Gall, D. V. DerVartanian, and H. D. Peck, Jr., Curt. Top. Bioenerg. 9, 237 (1979). 6 S. W. Ragsdale, L. G. Ljungdahl, and D. V. DerVartanian, J. Bacteriol. 155, 1224 (1983). 7 G. R. Bell, J.-P. Lee, H. D. Peck, Jr., and J. Le Gall, Biochimie 60, 315 (1978). 8 D. E. Stewart, J. Le Gall, I. Moura, J. J. G. Moura, H. D. Peck, Jr., A. V. Xavier, P. K. Weiner, and J. E. Wampler, Eur. J. Biochem. 185, 695 (1989). 9 j. Le Gall, Anal. Inst. Pasteur (Paris) 114, 109 (1968). 10 L. Chen, M.-Y. Liu, J. Le Gall, P. Fareleira, H. Santos, and A. V. Xavier, Eur. J. Biochem. 216, 443 (1993). J.-B. Park, F. O. Bryant, A. Shigetoshi, J. K. Magnuson, E. Eculston, J. B. Howard, M. F. Summers, and M. W. W. Adams, Biochemistry 30, 10885 (1991); P. R. Blake, J.-B. Park, F. O. Bryant, S. Aono, J. K. Magnuson, E. Eccleston, J. B. Howard, M. F. Summers, and M. W. W. Adams, Biochemistry 30, 10885 (1991)]; C.th, Clostridium thermosaccharolyticum [J. Meyer, J. Gagnon, L. C. Sieker, A. Van Dorsselaer, and J.-M. Moulis, Biochem. J. 271, 839 (1990)].

206

DISSIM1LATORY SULFATE REDUCTION

[14]

The first report of a physiological electron acceptor for D. gigas Rd has been published. ~ It is a rubredoxin-oxygen oxidoreductase (ROO), a homodimer of 43 kDa per monomer. The protein is a flavohemoprotein because it contains both FAD and a new type of heine group. Because the product of oxygen reduction by this protein is water, the following electron chain scheme has been proposed:

NADH

) NRO

Rd

2e + 02

> ROO

> H202 (slow)

4e + 02

>H20 (fast)

It is proposed that this electron transfer pathway is sufficient to explain the observation that ATP is formed from the degradation of polyglucose in the presence of oxygen.12 The similarity between the utilization of reduced pyridine nucleotides by D. gigas and the hydroxylation of hydrocarbons by Pseudomonas oleovorans is striking) 3 This could indicate that ROO may also play a role in a mixed oxygenase reaction that is still to be discovered. Another protein has been proposed to have an oxidoreductase activity toward Rd. This is the product of the rbo gene, called a rubredoxin oxidoreductase.~4,1~ This gene has been found in D. vulgaris Hildenborough. The protein, named desulfoferrodoxin, ~6 has no N A D H oxidoreductase activity. Because Rd is found in D. vulgaris Hildenborough ceils, which have no NRO activity, 9 more physiological roles for Rds remain to be discovered. Rubredoxin acts as an intermediate electron carder in the reduction of nitrates by Clostridium perfringens. 17 Such a function in strains of Desulfovibrio that are capable of the dissimilatory reduction of nitrates should

II L. Chen, M.-Y. Liu, J. Le Gall, P. Fareleira, H. Santos, and A. V. Xavier, Biochem. Biophys. Res. Commun. 193, 100 (1993). 12 H. Santos, P. Fareleira, A. V. Xavier, L. Chen, M.-Y. Liu, and J. Le Gall, Biochem. Biophys. Res. Commun. 195, 551 (1993). 13T. Ueda, E. T. Lode, and M. J. Coon, J. Biol. Chem. 247, 2109 (1972). 14 M. J. Brumlik and G. Voordouw, J. Bacteriol. 171, 4996 (1989). 15G. Voordouw, in "The Sulfate-Reducing Bacteria: Contemporary Perspectives" (J. M. Odom and R. Singleton, eds.), p. 88. Brock/Springer Series in Contemporary Bioscience, New York, (1993). 16 I. Moura, P. Tavares, J. J. G. Moura, N. Ravi, B.-H. Huynh, M.-Y. Liu, and J. Le Gall, J. Biol. Chem. 263, 21596 (1990). 17 S. Seki, A. Ikeda, and M. Ishimoto, J. Biochem. (Tokyo) 103, 583 (1988).

208

DISSIMILATORY SULFATE REDUCTION

[14]

"~-.~SG 42

FIG. 2. Stereo view showing the overall polypeptide fold of Desulfovibrio vulgaris rubredoxin, the Fe-Cys-4 complex, and the invariant residues listed in Fig. 1.

strained refinement procedure. 27 This allows a direct comparison with all the other rubredoxin crystal structures that were refined by similar techniques. Diffraction data of D. vulgaris Rd have subsequently been extended to 0.9 ,~ where the structural model has been refined by normal crystallographic (free atom) techniques (G. Sheldrick, private communication, 1994). This analysis is still in progress and is not considered here. We then compare the crystal structures of the different rubredoxins, taking into consideration structural features that appear to be important for maintaining the integrity of the rubredoxin molecule. The comparison of these heterologous proteins with their respective residue changes provides the possibility of determining some general rules concerning the structure of other rubredoxins for which the amino acid sequence has been determined and in mapping the recognition sites of NRO, ROO, and other redox partners. Figure 2 shows a stereo plot of the ~-C chain of the structure of the l-,~ model of D. vulgaris Rd, including the iron atom coordinated to the four-cysteine side chain groups and the invariant aromatic groups in the core of the molecule. Briefly described, the molecule contains fl-sheet structure, several 310 helical turns, and some glycine-type turns encompassing the aromatic dominated core. 2°-23 This specific core arrangement appears to be essential for the integrity of the metalloprotein structure and probably plays a role in controlling or stabilizing the redox states of 27 Z. Dauter, L. C. Sieker, and K. S. Wilson, Acta Crystallogr. Sect. B: Struct. Sci. B48, 42 (1992).

[14]

RUBREDOXIN IN CRYSTALLINE STATE

209

this type of metal center. The high variability in the sequence region 16 to 29 (flap of chain on the left in Fig. 2) is consistent with some of this polypeptide region missing in the "unique" D. desulfuricans Rd molecule. The crystal structures of all the Rds reported to date are similar, the only major changes in their tertiary structure being the deleted flap in D. desulfuricans Rd and one or two deletions in this region in other molecules and either a deletion at the N terminus (Pyrococcus furiosus Rd) or at the C terminus of some Rds. The structures can all be superimposed using the et-C atoms and the root mean square (rms) distance, for each residue from the reference molecule D. vulgaris Rd, can be calculated (see Fig. 3). An indication that the structures are similar is the low overall rms deviation between any two of them, on the order of 0.75 .~. A measure of the precision of the structure determinations is indicated by the values between the independently determined structural models of D. vulgaris Rd done at 1.0 and 1.5 ,~. It is encouraging to see that independent studies agree so well. At this point, it is difficult to judge the significance of the differences of 0.1-0.2 A in comparing these different structures. This becomes important in understanding the structural basis for the differences in redox potential of electron transfer proteins. C-x-y-C-G-z Chain Segments The amino acid sequences of all rubredoxins show two sets of the

-C-x-y-C-G-z- sequence around the iron center, where each cysteine is a ligand to the iron atom. The first set has significant changes at x and y, but z is maintained as a tyrosine residue. This aromatic residue appears to protect/maintain the F e - 4 C y s center relative to the interior of the molecule. The second set has the invariant Pro-40 in the x position. This strictly invariant proline appears to be in this location for a purpose. We suggest that it may be involved in an association with some part of the rubredoxin redox partner facilitating the exchange of the electron between the redox centers. Figure I shows a significant number of changes at y and a few changes at z. In addition, there is always an aromatic residue two residues before the first cysteine of either -C-x-y-C-G-z- chain segment. This aromatic residue can be a tyrosine, phenylalanine, and tryptophan at position 4, which is somewhat near the surface of the molecule. The equivalent residue, in the second chain segment, is an invariant tryptophan at position 37. This tryptophan is located toward the interior of the molecule and is close to the F e - 4 C y s center and is in association with the aforementioned tyrosine at position 11 in the polypeptide chain. It appears to protect or poise the iron center for its activity. The aromatic residue at position 4

210

DISSIMILATORY

8

SULFATE

4

I

I

D v R d ( 1 . 5 A resolution) - DgRd DdRd ......

3.5

A

[14]

REDUCTION

E o 3 2.5

"0

n>

o E U eo~

2

A 1.5 1

L'

..

/t

0.5 ~ : , I '~'

:'.

,

.-..:,

...

,"

.

,.; . ":~~

:

,

,,, t

,'

."

~ ~ /",,

~' ' ". : ' .: %~ , ,,Y

,/"J

;

~' ,

, ~1 ~t

L

0 0

b ¢/)

10

20

30 40 Residue Number

50

60

4 CpRd - PfRd (oxidized) . . . . . PfRd (reduced) ......

3.5

E =o 3

v "13 rr

E

2.5

2 !

1.5 H !

"'..

H ! k5

' ',',

0 i

0

~,,

" ,

i

i

20

,i/

~!l

,-'", :'.

10

i/

;

i

I,'

i

30 40 Residue Number

i

50

60

FIG. 3. Root mean square differences between backbone atoms of the rubredoxin crystal structures after superposition of the c~-carbon atoms on D. vulgaris Rd. (a) Desulfovibrio rubredoxins. (Note the large differences associated with the residues bridging the deleted segment in D. desulfuricans Rd.) (b) Mixed bacterial rubredoxins.

[14]

RUBREDOXIN IN CRYSTALLINE STATE

211

can be variable, in accordance with its location near the surface of the molecule, but the tryptophan is strictly invariant, being located in the interior of the molecule and somewhat closely associated with the iron center. Of the 13 residues that are strictly invariant, the two -C-x-y-C-Gsegments account for 6 of these residues. In addition, there are five aromatic residues, one proline, and one lysine that are conserved.

Invariant Lys-46 Lys-46 presents an interesting situation for discussion. From the sequence comparisons it is shown to be the only strictly invariant hydrophilic residue in all the Rds. Except for the D. oulgaris Rd structure, the crystal structures of the other four Rds show that Lys-46 extends across to the neighboring chain, making an H bond to the carbonyl oxygens of residue 30 and residue 33, presumably contributing to the stability of the molecule. This is not the situation in the crystal structure ofD. vulgaris Rd, in which the side chain of Lys-46 bends around to make an H bond with N46 and with an oxygen atom of a sulfate ion, which in turn makes an H bond to N47 of the main chain. It seems clear that this invariant residue does more than stabilize the molecule.

Structure and Redox Function There are several structural features that have been suggested as playing a role in modulating the redox properties of the metal center. These include the metal complex itself, the NH--S hydrogen bonds of the protein residues with the metal complex, and some of the aromatic side chains in the core of the molecule. ~8'28No major differences have been found in any of these features. This is not surprising because the differences in redox potential among the different Rds are rather modest. On the other hand, these same features indicate what is necessary to maintain the rubredoxin structure and the general range of redox potentials. The structure of D. desulfuricans Rd indicates that residues 20-26 of the chain are not necessary to maintain a stable and functional Rd. Pyrococcusfuriosus Rd shows a tryptophan in place of tyrosine or phenylalanine at position 4, but this is at the opposite end of the molecule where somewhat larger structural adjustments can be accommodated without 2a E. T. Adman, K. D. Watenpaugh, and L. H. Jensen, Proc. Natl. Acad. Sci. U.S.A. 72, 4854 0975).

212

DISSIMILATORY SULFATE REDUCTION

[14]

disrupting the integrity of the molecule. Figure 4 shows the superimposed structures for the aromatic side chains in this region. Day et al. 22 point out that the additional H bond supplied by this tryptophan residue probably contributes to the thermal stability of P. f u r i o s u s Rd. Fe-4Cys Center Small differences between the molecules are also seen when comparing the bond lengths and angles of the metal center (see Table I). The differences seen in the Fe S bond distances show a trend but, owing to the target values and the restraints used in the refinement of these crystal structures, the accuracy of these individual values should not be overemphasized. On the other hand, subject to the decisions made on target values and restraints, the average of these distances may have some significance relative to the individual values. Although the spread of the Fe S bond distances is relatively large among the oxidized structures, it is interesting to note that the average distance of the symmetry-related pair Fe-SG6 and Fe-SG39 is about 2.30 ~ compared to an average distance of about 2.26 A for the pair Fe-SG9 and Fe-SG42. Although the accuracy of these values can be questioned they do show that the twofold symmetry is maintained around the iron atom and that the Fe---S bond distances of the bonds closer to the center of the molecule are longer than those to the exterior. Adman et al. pointed out that there are two NH--S hydrogen bonds to SG6 and SG39 but only one to SG9 and SG42. 28 The NH--S

FIG. 4. Stereo view of the aromatic side chains at position 4 and the immediate core environment, showing the positional variability of the superimposed structures.

[14]

RUBREDOXIN IN CRYSTALLINE STATE

213

TABLE I BOND LENGTHS AND ANGLES OF Fe-4Cys CENTER

Center

8RXN

7RXN

1RDG

6RXN

4RXN

Ave. oxidized ~

5RXN

1CAA

ICAD

2.32 2.29 2.30 2.25

2.32 2.25 2.33 2.25

2.34 2.29 2.35 2.29

2.31 2.27 2.30 2.26

114.3 109.3 104.0 103.9 113.6 111.9

113.0 111.8 102.6 102.4 115.2 112.3

112.6 113.4 104.6 102.7 111.2 112.7

113.6 110.9 104.9 103.2 111.9 112.4

3.51 3.58 3.47 3.42 3.52 3.49

3.34 3.43 3.41 3.36 3.47 3.50

3.55 3.58 3.48 3.54 3.60 3.60

Bond distances (.~) Fe-SG6 Fe-SG9 Fe-SG39 Fe-SG42

2.29 2.26 2.29 2.26

2.33 2.29 2.29 2.27

2.32 2.29 2.28 2.27

114.8 110.9 105.8 104.5 109.2 111.8

113.8 110.2 106.2 104.0 109.6 113.2

114.5 111.3 106.1 103.4 109.8 111.9

2.28 2.26 2.30 2.25

2.34 2.29 2.31 2.23

Bond angles (degrees) SG6-Fe-SG9 SG6-Fe-SG39 SG6-Fe-SG42 SG9-Fe-SG39 SG9-Fe-SG42 SG39-Fe-SG42

111.4 112.8 104.7 100.8 114.1 113.4

113.8 108.6 104.1 103.7 114.4 112.4

NH--S hydrogen bonds distances (/~) SG6--N8 SG6--N9 SG9--Nll SG39--N41 SG39--N42 SG42--N44

3.54 3.55 3.42 3.58 3.62 3.50

3.56 3.53 3.47 3.57 3.64 3.41

3.53 3.54 3.53 3.59 3.64 3.49

3.49 3.61 3.54 3.51 3.56 3.86

3.63 3.67 3.44 3.55 3.72 3.90

3.67 3.67 3.46 3.58 3.61 3.84

The unconstrained 4RXN is not included in the averages. b C. pasteurianum Rd, 4RXN, 1.2 ,~ [Watenpaugh et al., 1979 (unconstrained model)21; C. pasteurianum Rd, 5RXN, 1.2 ,~ (K. D. Watenpaugh (unpublished work; 1984); D. gigas Rd, IRDG, 1.4 .A (Frey et al.)lS; D. desulfuricans Rd, 6RXN, 1.5 ~ (Stenkamp et a/.)19; D. vulgaris Rd, 7RXN, 1.5 A (Adman et al.)2°; D. vulgaris Rd, 8RXN, 1.0 ,~ (Dauter et al.)27; P. furiosus Rd, 1CAA, 1.8 ,~ (Day et al.):2; P. furiosus Rd, 1CAD, 1.8 ,~ (Day et al.22)]

bonds of SG6 to backbone amides of 8 and 9 and the one from SG9 to N 11 are on one side of the twofold axis of symmetry whereas the NH-S bonds of SG9 to the amides at positions 41 and 42 and of SG42 to position 44 are on the other side. The SGs with the two NH--S bonds have the longer Fe---S bond length whereas the SGs with the single NH--S bond have the shorter Fe S bond length. Confirmation of this interesting pattern of NH--S bonds above and below the twofold axis of symmetry requires additional higher resolution structure analyses. Considerable variation is seen in the NH--S hydrogen bond distances among the individual oxidized Rds. Relative to the local twofold axis of

214

DISSIMILATORY SULFATE REDUCTION

[14]

symmetry, the average values of these distances (see Table I) in the oxidized molecules show no obvious symmetry. The average distance is about 3.56/~. Figure 5 shows a stereo view of the twofold configuration around the iron center in D. vulgaris. Reduced Rubredoxin

Because the reduced Pf structure has essentially the same metal site geometry as the oxidized forms, it appears that only small structural changes occur with the change in oxidation state. In these comparisons of the oxidized versus reduced models, the lower resolution of the P. furiosus Rd structures is the limiting factor. The Fe---S bonds of the reduced molecule appear to be about 0.03 A longer than the averages of the oxidized Fe--S bond distances. The average of NH--S bond distances of the reduced P. furiosus Rd is 3.42/~ and is therefore 0.14/~ less than the average of the oxidized forms. Within the precision of the current values of the bond lengths for the different Rds, this decrease in the NH-S distance on reduction of P. furiosus Rd is probably significant. Specificity Figure 6 shows the charged side chains on the surface of the Rd molecules. Most of the variability in residue distribution occurs at the opposite end of the protein from the iron center (bottom of this stereo

I

-"

l~

,41

FIG. 5. Stereo view of the Fe-4Cys center, showing the pseudotwofold symmetry and the NH--S hydrogen bonds.

[14]

RUBREDOXlN IN CRYSTALLINE STATE

215

Fro. 6. Stereo view of the charged side chains relative to the hydrophobic Fe-4Cys center.

view). The iron center is located in the molecule in a region surrounded by hydrophobic residues. The external residues are the most likely candidates for specificity interactions with its redox partners. As mentioned above in the discussion of the physiological role of the Rds the D. gigas Rd exchanges electrons very well with the NRO from D. gigas but D. vulgaris Rd does not. 9,1° Obviously some differences in the molecules are responsible for this change in electron transfer. The iron center is in a rather invariant hydrophobic environment at the opposite end of the molecule from the variable charged residues. A reasonable proposal is that the invariant hydrophobic region provides the common docking and electron exchange region whereas the more variable region of Rd provides the specificity to interact with its redox partner. The Rd molecule is sufficiently small that this should not require an extensive docking region of its much larger redox partner. The variable residue at position 7 could play a special role in docking or the electron exchange. Desulfooibrio gigas Rd is distinct from D. oulgaris Rd in several ways, as shown in Fig. la. It is interesting to note that, among the 13 Rds, only D. gigas Rd has residue 3 as isoleucine in place oflysine. This hydrophobic surface residue is probably contributing to some of the specificity of this particular protein. Conclusion The high-resolution crystal structures of five different rubredoxins show that these iron proteins have a strong conservation of the stereo-

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[15]

chemical geometry around the Fe-4Cys center. Although more flexible, the arrangement of the aromatic core is conserved. The polypeptide chain folds into a rather rigid configuration contributing to the stability of the core residues and the iron center. In spite of the small number of amino acids, the molecule shows a large diversity in the surface residues. These surface residues likely provide the specificity for docking the most invariant hydrophobic portion of the molecule, which contains the Fe-4Cys redox center. At the current state of analysis of several structural models (1.0 to 1.8 A) two new features seem to be apparent. The cysteinyl sulfurs with two NH--S bonds appear to have longer Fe---S bonds than the cysteinyl sulfur with one NH--S bond. An average decrease of about 0.17 ,~ in the NH--S hydrogen bond distance occurs when the Fe-4Cys center goes from the oxidized state to the reduced state. Acknowledgments We acknowledgethe use of Molscript29for Figs. 2, 4, 5, and 6. X-Ray structures are from the BrookhavenProtein Data Bank)°,31 29p. j. Kraulis,J. Appl. Crystallogr. 24, 956 (1991). 30F. C. Berstein, T. F. Koetzle, G. J. B. Williams,E. F. Meyer, Jr., M. D. Brice, J. R. Rodgers, O. Kennard, T. Shimanouchi,and M. Tasumi,J. Mol. Biol. 112, 535 (1977). 31E. E. Abola, F. C. Bemstein, S. H. Bryant, T. F. Koetzle, and J. Weng, in "Crystallographic Databases--Information Content, Software Systems, Scientific Applications" (F. H. Allen,G. Bergerhoff,and R. Sievers,eds.), p. 107.InternationalUnionofCrystallography, Bonn/Cambridge/Chester,1987.

[15] C h a r a c t e r i z a t i o n o f T h r e e P r o t e i n s C o n t a i n i n g M u l t i p l e I r o n Sites: R u b r e r y t h r i n , D e s u l f o f e r r o d o x i n , a n d a P r o t e i n Containing a Six-Iron Cluster By

ISABEL

MOURA, P E D R O

TAVARES,

and

NATARAJAN RAVI

Introduction It has been demonstrated that a number of proteins isolated from anaerobic sulfate reducers contain many novel combinations of iron centers. The unusual and diversified metal centers found in these proteins are more than a surprise, as no one could have envisaged the possibility of the existence of such metal centers, and needless to mention they offer METHODS IN ENZYMOLOGY, VOL. 243

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