Protein determinants of metal site reduction potentials: site-directed mutagenesis studies of Clostridium pasteurianum rubredoxin

ELSEVIER

Inorganica Chimica Acta 242 (1996) 245-251

Protein determinants of metal site reduction potentials: site-directed mutagenesis studies of Clostridium pasteurianum rubredoxin’ Qiandong Zenga, Eugene T. Smithb, Donald M. Kurtz, Jr.a, Robert A. Scotta,* aCenterfor Metalloenzyme Studies, University of Georgia, Athens, GA 30602-2556, USA bDepartment of Chemistry, Florid0 Institute of Technology, Melbourne, FL 32901-6988, USA

Abstract Site-directed mutagenesis of Clostridiwn pusteurianum

has been used to study the effects of mutations resulting in surface charge changes near the Fe(Cys), site rubredoxin (Rd). As predicted by simple electrostatics considerations, Rd variants with positively charged

arginine residues in place of neutral surface residues ([V8R] and [LAlR]) exhibit significant increases in the Fe(II/III) reduction potential. Contrary to electrostatics predictions, [V8D] and [V41D] Rd variants also exhibit significant increases in the Fe(II/III) reduction potential. These results indicate that protein electrostatic effects do not dominate as determinants of metal-site reduction potential in C. pusteurianum Rd. A hypothesis is developed that increased solvent accessibility and the resultant increase in polarity of the Fe(Cys)4 site dominate as determinants of the reduction potential in this protein. Possible experimental tests of this hypothesis are discussed. Keywonis:

Metalloprotein; Iron-sulfur protein; Protein electrochemistry

1. Introduction The ability of the polypeptide to tune the differential stability of oxidized and reduced forms of redox cofactors has long been recognized as the key to evolution of electron-carrying proteins and redox enzymes. However, the molecular interactions that are used to accomplish this redox tuning have remained elusive. Because of the expected charge cycling of the redox cofactors, alteration of electrostatic characteristics of the protein environment has received much attention as a possible contributor. For example, a study of reduction potential changes in surface charge variants of cytochrome b5 [l] and Azotobacter vinelundii ferredoxin [2] have been discussed in terms of electrostatic effects, and several studies of cytochrome c have suggested the importance of such effects (e.g. [3,4]). Another popular candidate for redox tuning involves dipolar (as opposed to monopolar) charge effects, which can be variously described as polarity or hydrophobic* Corresponding author. Tel.: +l 706 5422240; fax +l 706 5429454; e-mail: [email protected] ’ This paper is dedicated to Professor Harry B. Gray.

0020-1693/96/$15.00 Q 1996 Elsevier Science S.A. All rights reserved SSDI 0020-1693(95)04874-7

ity/hydrophilicity, and can be affected by solvent accessibility. For example, solvent exposure has been suggested to affect reduction potentials of heme proteins [571 as well as Fe-S proteins [&lo]. We have chosen to develop rubredoxin as a prototypical metalloprotein for mutagenesis studies targeted at understanding protein determinants of reduction potentials. Rubredoxins (Rds) are the smallest (M, = 6000) and simplest of redox metalloproteins and have been identified from at least 11 species [ 11,121. They generally contain a single [Fe-@-Cys),] site (with the exception of Pseudomonas oleovorans Rd, which has two such centers [13]) in a C-X2-C-X21_2s-C-X2-Camino acid sequence motif. To date, five X-ray crystal structures and one NMR structure have been reported for Rds and all of these Rds have a strikingly similar main-chain tertiary structure [ 12,141. Reduction potentials range from -60 mV for Rd from Chlorobium limicolu f.sp. thiosulfatophilum [15] to +42 mV for Rd from Megusphuera efsdenii [161. The Rdlike center in the non-heme iron-sulfur protein rubrerythrin (contained in a C-X2-C-Xt2-C-X*-C motif) has a reduction potential of +230-260 mV [ 17,181 (Table 1). Thus, comparisons of the redox chemistry among various

Chimica Acra 242 (1996) 245-251

Q. Zeng et al. /Inorganica

246

Rds can potentially provide insight into determinants of metalloprotein reduction potential. However, the global amino acid sequence variations and subtle structural differences among Rds make it difficult to identify specific factors controlling the redox thermodynamics. A survey of soluble one-electron carrier proteins indicated that lower potential proteins tend to be more negatively charged [19]. In agreement with this observation, removal of an internal positive charge in cytochrome c [20] or addition of an internal negative charge in myoglobin [21] causes a lowering of the reduction potential. However, recent examples reveal that the effect of mutations resulting in a change of the net charge is more complicated. In a recent study of flavodoxin, six negatively charged surface residues were systematically neutralized by changing aspartate or glutamate to asparagine or glutamine, respectively, and mixed results were obtained [22]. For the oxidized flavin/semiquinone couple, the reduction potential became slightly more negative with a very weak correlation between the number of charges neutralized and the reduction potentials, while for the semiquinonelhydroquinone couple, the reduction potentials were shifted to more positive values and were largely additive with the number of charge changes. In Peptococcus aerogenes ferredoxin, only cluster I is surrounded with a cluster of negative charges yet the reduction potentials for clusters I and II are almost identical [231. In a study of Azotobucter vinelandii ferredoxin I, changing Asp-15 near the [3Fe-4S] cluster to asparagine increases the reduction potential of the [3FeAS] cluster by 20 mV [24], while substitution of negatively charged

surface residues with neutral residues (D23N, E38S, E46A) or substitution of a histidine with a negatively charged residue (H35D) had little or no effect on the reduction potential [2]. Jensen et al. concluded that the charges of the ionized and protonated groups do not seem to contribute significantly to the reduction potentials of the [Fe-S] clusters in ferredoxins [8]. Rubredoxins are generally highly negatively charged at neutral pH. Table 1 lists the amino acid sequences, the reduction potentials and the net peptide charges of the nine rubredoxins and the Rd-like domain of rubrerythrin for which reduction potentials have been reported. (Effectively, the net peptide charge corresponds to the overall charge expected on the apoprotein.) There seems to be some correlation between the net charges and the Fe3+/2+ reduction potentials. As would be predicted from simple electrostatics arguments, reducing the net negative charge leads to a more positive reduction potential (i.e. it becomes ‘easier’ to add an electron to Fe3+). Given these observations, we decided to undertake a systematic investigation of the effect of charges near the [FeCys4] site of Clostridium pasteurianum rubredoxin on its reduction potential. We report herein the effect of site-directed mutagenesis of residues near the Fe site on the reduction potential of Clostridium pasteurianum Rd. Fig. 1 shows schematically the locations of the residues subjected to mutagenesis in this study. Specifically, we have constructed and expressed genes for the following C. pasteurianum Rd variants: [V8R], [L41R], [V8R, UlR], [V8D], [L41D] and [T5R, TYR]. Val-8 and Leu-41 were selected because

Table 1 Amino acid sequence alignments, redox potentials, and net peptide charges of rubredoxins Rubredoxi#

Amino acid

and rubredoxin-like

domains

.?P WV, NHE)

sequenceb 1 0

1

2 0

3 0

4 0

5 0

Chl. limit.

MQKYVCISV~G

YVYDPADGEP

DDPIDPGTGF

EDLPEDWVCP

VCGVDKDLFE

Cl. past.

MKKYTCTVCG

YIYNPEDGDP

DNGVNPGTDF

KDIPDDWVCP

L.CGVGKDQFE EVEE

B. methyl. Ps. oleov.

41

-57

Ref.

-12.0 -9.0

WI r351

MQKYVCDIM

YVYDPAVGDP

DNGVAPGTAF

ADLPEDWVCP

ECGVSKDEFS

PEA

-40

-9.0

[%I

xYLKWI_CITcG

HIYDEALGDE

AEGFTPGTRF

EDIPDDWCCP

XGATKEDYV

LYEEK

-37

-9.5

AKWVCKICG

YIYDEDAGDP

DNGISPGTKF

EELPDDWVCP

ICGAPKSEFE

KLED

MKKYVCTVCG

YEYDPAEGDP

DNGVKPGTSF

DDLPADWVCP

VQGAPKSEFE

AA

MKKYWl’V~G

YEYDPAEGDP

DNGVKPGTAF

EDVPADWVCP

ICGAPKSEFE

PA

+5

-6.0

MDIYVCTVCG

YEYDPAKGDP

DSGIKPGTKF

EDLPDDWXP

VCGASKDAFE

KQ

+6

MDKYECSIM

YIYDEAEGD.

DGNVAAGTKF

ADLPADWVCP

TCGADKDAFV

KMD

-6.0 -8.0

xATKWR!2RNCG

YVHEGT:GAP

. . . . . . . . . .

.EL.....CP

WPKAHFE

LLGINW

[131 1371 [I71 [381 [391 WI U61 [171 1181

Py.JiAriosus D. vulg. (H) D. vulg. (M) D. gigas M. elsdenii D. vulg. Rr

PES

Net peptide charged

/

0 0

+23 +42 +230 +260

-8.0 -6.0

+2.5

Vhl. limit., Chlorobium limicola

f. sp. rhiosulfarophilum; Cl. past., Closwidium pasteunkuwm; B. merhyl., Butyribac~erium methylotrophicum; Ps. oleov., Pseudomonas oleovorans; Py fwiosus, P~IVCOCCUS fin’osus: D. vulg. (H). Desulfovibtio vulgaris. strain Hildenborough; D. vulg. (&I), Desulfovibrio vulgaris,strain Miyazaki; D. gigas, Desulfovibriogigos; M. elsdenii,Megasphaeraelsdenii; D. vulg. Rr, Desulfovibn’o vulgaris rubrerythn’n.

Closkdium posreurionummbredoxin is used throughout. x at the beginning of the sequence Cysteine ligands are underlined. ‘Midpoint oxidation/reduction potential. in millivolts versus the normal hydrogen electrode. dNet charge of the apoprotein calculated at neutral pH (-1 for D, E, C-terminus; +l for K, R, N-terminus; +0.5 for H). bathe numbering

system for

sequence that is unspecified

here.

indicates

extra upstream

Q. Zing et al. /Inorganica

Fig. 1. Placement of wild-type C. pasteurianum mbredoxin amino acid residues that have been substituted using site-directed mutagenesis in this study. Thr-5, Thr-7, Val-8, and Leu-41 were substituted by the residues indicated. The FeS4 site is indicated in space-filling format. This figure was generated with MolScript [41].

they are the two non-ligating surface residues nearest to the iron center. These two neutral residues were substituted by positively charged (arginine) or negatively charged (aspartic acid) residues. Arginine was preferred to lysine since the positive charge of lysine tends to be more highly solvated [25]. We also constructed a [T5R,T7R] variant, based on the hypothesis that the two arginine residues flanking one of the cysteine ligands in the Rd-like domain of rubrerythrin (Table 1) may be responsible for its much higher reduction potential. The mutant and wild-type Rd genes were expressed in E. coli, and the overexpressed proteins were purified, characterized, and their reduction potentials measured. 2. Experimental

2.1. PCR-assisted site-directed mutagenesis of the C. pasteurianum Rd gene Molecular biology procedures generally followed those described in [26] or in Current Protocols in Molecular Biology [27]. C. pasteurianum Rd mutant genes were generated using polymerase chain reaction (PCR)-assisted site-directed mutagenesis. The template was a synthetic gene for C. pasteurianum Rd [28], corrected to the recently published wild-type sequence [29]. All PCR primers were synthesized by Integrated DNA Technologies, Inc. N-terminal primers for substitutions at positions 5, 7, and 8, were designed with NdeI sites (CATATG) incorporating the start codon and generally contained five to seven bases beyond the site of mutation. (An exception was the N-terminal primer for the [T5R,T7R] mutation which contained 21 bases beyond the R7 codon.) Cterminal primers for substitutions at position 41 were de-

Chimica Acta 242 (I 996) 245-2.51

247

signed with Hind111 sites (AAGCTT) incorporating the last two bases of a second stop codon [28] and generally contained five or six bases beyond the site of mutation. For PCR amplification, the protocol recommended by Perkin-Elmer was modified for ‘hot-start’ PCR. Briefly, the wild-type C. pasteurianum Rd gene was inserted between the NdeI and Hind111 sites of pTi’-7 [30]. This plasmid was linearized by BgflI (Boehringer-Mannheim) digestion and used as the template, which was mixed with primers and incubated for 5-10 min at 94°C for ‘hot-start’ PCR. Then a mixture containing the appropriate volumes of dATP, dCTP, dGTP, dlTP, 10X PCR buffer, and Taq polymerase was added to the template-primer mixture to initiate the PCR. Amplification was achieved using 30 of the following temperature cycles: 94°C for 1 min, 37°C for 1 min, 72°C for 2 min. The resulting PCR product was purified with Wizard PCR-preps (Promega) and then mixed with purified pT7-7. This PCR product/plasmid mixture was double-digested with NdeI and Hind111 (Boehringer-Mannheim), the DNA purified by phenol extraction and ethanol precipitation, and then ligated with T4 DNA ligase (Promega). The ligation product was used to transform E. coli strain 71/18 and positive colonies were identified by mini-prep and HpaI and/or KpnI (United States Biochemical) digestion of the plasmid (HpaI and KpnI cut the C. pasteurianum Rd gene, but do not cut the vector). Plasmids from positive colonies were purified by Midi-Prep (Qiagen). The nucleotide sequence of the inserted gene was confirmed by DNA sequencing at the University of Georgia Molecular Genetics Instrumentation Facility. The plasmid containing the mutant gene was then used to transform E. coli strain BL21(DE3) (Novagen) for expression of the mutated proteins. 2.2. Overexpression and purification of mutated proteins E. coli strain BL21(DE3) containing either wild-type or mutant plasmid was grown with shaking (250 rpm) at 37°C in 2 1 Luria-Bertani (LB) medium supplemented with ampicillin (lOOpg/ml), until the optical density at 600 nm reached 1-1.3, at which time IPTG was added to a final concentration of 0.4 mM. The cultures were shaken for three more hours, then centrifuged for 10 min at 5000 X g, the medium discarded, and the cell pellet resuspended in 50 mM Tris buffer (pH 7.5), containing 2 mM EDTA. The overexpressed protein was released from the cells by sonication or through repeated freeze/ thaw cycles [31]. The lysate was centrifuged at 27 000 X g for 30 min and the supernatant was loaded onto a 20-ml QAE-Sephadex G-25 (Pharmacia) column. This column was washed extensively with 100 mM NaCl in 50 mM Tris buffer (pH 7.5). A red band appearing at the top of the column was eluted by washing with 500 mM NaCl in the same Tris buffer. This red fraction was further purified by ultrafiltration in an Amicon cell (Amicon) with a 3000 MWCO membrane and by prepa-

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248

rative FPLC separation on Mono-Q (Pharmacia). Preparative and analytical separations were performed on Mono Q HR IO/10 and HR 5/5 columns, respectively, connected to a Pharmacia FPLC system which consists of a GP-250 gradient programmer, two P-500 high precision pumps, a UV-1 single-path monitor, a V-7 injection valve, and a REC-482 two-channel chart recorder. The ionic strength gradient was generated using different proportions of buffer A (50 mM Tris buffer, pH 7.5) and buffer B (1 M NaCl in buffer A), both filtered through 0.22~pm membranes. A typical gradient for the wild-type C. pasteurianum Rd consists of a linear gradient from O20% B in 5 ml, from 20-30% B in 60 ml, and from 30100% B in 2 ml. The peak containing the red protein (the Fe form of Rd) was collected, concentrated, desalted in an Amicon cell, and stored in 10 mM sodium phosphate buffer (pH 7.5) at -80°C.

visible spectra were recorded on a Shimadzu UV-21OlPC scanning spectrophotometer, using quartz cuvettes ( 1-cm light path). EPR spectra were recorded at liquid helium temperature on a Bruker ESP-300E spectrometer equipped with an ER-4116 dual mode cavity and an Oxford Instrument ESR-9 flow cryostat. 2.4. Molecular modeling Energy-minimized structures of mutated C. pasteurianum Rd were generated from the energy-minimized X-ray crystal structure of the wild-type protein (PDB code Srxn) on a Silicon Graphic9 workstation using the SYBYL package (Tripos). Solvent accessible surfaces were also calculated with SYBYL using a spherical water radius of 1.4 A. 3. Results and discussion

2.3. Characterization of mutated proteins Cyclic voltammograms were obtained utilizing a micro-electrochemical cell in a three-electrode configuration as previously described [32]. The working, counter, and reference electrodes were edge-plane pyrolytic graphite, platinum, and Ag/AgCl, respectively. Current/potential data were recorded with a BAS CV-50 W voltammetric analyzer. A 20-~1 sample of 100,~M protein in 25 mM sodium phosphate buffer (pH 7.5), containing 150 mM MgC12 as the supporting electrolyte. The peak current for most variants was directly proportional to the square root of the scan rate from 2 to 200 mV s-l, indicating that the electrode response was diffusion controlled and rapid. Midpoint potentials were calculated by averaging the anodic and the cathodic peak potentials and peak separations ranged from 60 to 76 mV (cf. Table 2), which was slightly greater than the theoretical value of 59 mV. UVTable 2 Reduction potentials of mutated C. parteutianum rubredoxins Rd Variant

E”’ (mV) NHE (AEp)’

Al? (mV)b

Wild-type [TSR, ‘K’RI

-55 (74) -50 (62) -15 (68) +3 (60) +30 (60) -28 (67) -23 (76)

+5 +40 +58 +85 +27 +32

[V8Rl [LAIRI [V8R, IAIR]

rV8Dl [J-4lDl

Distance(s) (AP 10.0, 10.9 8.5 7.4 8.5.7.4 4.7-6.Od 5.5-6.5d

a E”’ is the midpoint and AE,, is the separation of the cathodic and anodic peaks of the cyclic voltammogram. bChange in reduction potential from wild-type. CDistance of the charged group of the mutated residue (arginine guanidino or aspartic acid carboxylate) from Fe in the Sybyl energyminimized variant structure. dFor the aspartic acid variants, this is the range of distances spanned by the two carboxylate oxygens.

Our choices for substitution of C. pasteurianum Rd residues were dictated mainly by their proximity to the Fe(Cys),,site. Thr-5, Thr-7, Val-8, and Leu-41 constitute the set of non-conserved residues which are adjacent to one of the Cys ligands (positions 6, 9, 39, 42) (Table 1). We have not attempted substitution of the conserved residues Gly-10, Gly-43, Pro-40, or Val-38 in order to avoid possible disturbance of the Fe(Cys), site structure. In addition, SYBYL calculations indicate that the solvent accessible surface areas of the side chains of Val-8 and Leu-41 are 43 (of 150 [33]) and 57 (of 170) A*, or 28% and 34% of the total available side-chain surface, respectively. In contrast, the solvent accessible surface areas of the Val-24 and Val-38 side chains are 12% and 25%, respectively. Therefore, the side chains of Val-8 and Leu-41 are relatively highly exposed to solvent and substitution of these residues with charged residues should not cause significant disturbance to the folding and conformation in general and the iron center in particular. UV-visible and electron paramagnetic resonance (EPR) spectroscopic characterization of the variants confirms this prediction (vide infra). The net charges of these C. pasteurianum Rd variants were probed by anion-exchange chromatography (FPLC Mono Q). Fig. 2 shows an elution profile of an approximately equimolar mixture of wild-type, [VSR], [L41D], and [V8R, LAlR] variants, each of which should have a different net charge. The relative elution position indicates that the surface charge of Rd has been altered as expected for all variants. The anion-exchange elution profile provides a simple and convenient way to characterize these ‘charge-change’ variants and corroborates plasmid sequencing results confirming that the mutagenesis was successful. W-visible and EPR spectra indicate that the electronic structure of the iron site is not significantly affected in either oxidation state by these charged mutations. Fe(III)

Q. Zeng et al. /Inorganica Chimica Acta 242 (1996) 245-251

I

0

10

I

20 30 40 50 Elution Volume (mL)

I

600

Fig. 2. Anion exchange elution profile of mixture of four C. pasteurianum Rd variants, exhibiting the expected charge differences. The mutations are indicated above each elution peak and the net peptide charge (calculated as detailed in Table 1) is given in the circular label on each peak. The contents of buffers A and B a~ given in Section 2.

forms of [LAlD] and [V8R, IAlR] show the characteristic UV-visible maxima at 492, 379, 277 nm, while maintaining an AzsdAdm ratio of 2.3-2.4; these values are virtually identical to those of wild-type C. parteurianum Rd (Fig. 3). UV-visible spectra of Fe(I1) forms of these variants are also virtually identical to those of wild-type Rd (Fig. 3, inset). Similar results are obtained for all the other variants discussed herein (data not shown). The EPR spectra of Fe(II1) forms of [L41R] and [LAlD] variants are also very similar to that of wild-type C. pasteuriunum Rd, exhibiting g values of 9.6 and 4.3. The slight differences in g = 4.3 EPR signals (most obvious in the ‘wings’) are most likely due to slightly altered microheterogeniety of the sample (‘g-strain’). Again, similar results are obtained for all the variants (data not shown). Overall, the insignificant changes observed in the thiolate + Fe(II1) charge-transfer transitions, the EPR g values, and rhombicity all suggest that the electronic properties of the Fe(II1) sites are essentially unchanged in these variants. The charge-transfer region of the Fe(I1) Wvisible spectra suggest that the reduced forms also exhibit essentially unchanged electronic properties. Simple coulombic arguments predict that introduction of a positive charge near the Fe(Cys), site would raise the reduction potential of this site and that the closer the positive charge resides, the larger the potential shift would be. The reduction potentials measured for the arginine variants seem to follow this trend (Table 2). The

249

[T5R, T7R] variant contains two new positive charges ca. 10 8, from the Fe (as estimated from the energyminimized model structure for this variant) but exhibits only a small increase (+5 mV) in reduction potential. (This result suggests that something other than these positively charged residues is responsible for the high reduction potential of D. vulgaris rubrerythrin (Table l).) As predicted, more significant increases in the reduction potential obtain when the nearer residues V8 and LA1 are replaced with R: [V8R] causes a +40 mV shift and [L41R] a +58 mV shift, which are qualitatively consistent with estimated distances of the arginine guanidino group from the Fe of 8.5 and 7.4 A, respectively (Table 2). Also, these effects are approximately additive with a reduction potential shift of +85 mV measured for the double variant [VSR, L41R]. Extension of these coulombic arguments would predict a lowering of the reduction potential upon substitution with a negatively charged residue. The measured reduction potentials for the [V8D] and [L41D] variants do not bear out this prediction (Table 2). Increases in the reduction potential are observed for both variants with substitution at position 41 again having a slightly larger effect than substitution at position 8. The overall effect of aspartic acid incorporation appears to be somewhat smaller than the effect of arginine incorporation. Assuming that these reduction potentials measured at a single pH and ionic strength are representative, the inescapable conclusion from these comparisons is that the net charge of amino acid side chains alone cannot explain the shifts in reduction potentials in these C. pasteurianum Rd variants. It is instructive to consider solvent accessibility (and resultant changes in local dielectric constant or polarity) of the Fe(Cys), site as an alternative to electrostatics as the dominant effect giving rise to the reduction potential shifts shown in Table 2. Increased solvent accessibility of

I

I

I

300

400

500

-...__ --....__..___ _...___.__........._.... -..________ _._..._ I 600 700 I )O I

1 Mm) Fig. 3. UV-visible spectra for representative Fe(lII) and ascorbatereduced Fe(I1) forms (inset) of C. pasteurianum Rd variants: wildtype (solid); [VIR,LAlR] (long dash); and [UlD] (short dash). The similarity of these spectra implies the absence of significant changes to the Fe(S-Cys)e electronic st~ctunz. as judged by the position and intensity of charge-transfertransitions. The spectra have been offset vertically for clarity.

Q. Zeng et al. /Inorganica Chimica Acta 242 (1996) 245-251

I

1000

I

I

2000 3000 Magnetic Field (Gauss)

I

4000

Fig. 4. Electron paramagnetic resonance (EPR) spectra of representative C. pas~eurianum Rd variants: (a) [LAlD]; (II) wild-type; (c) [LAIR]. The feature near 3300 G (g = 2) in (b) was associated with the EPR cavity. The small differences in shape of the g = 4.3 resonances are likely related to variations in microheterogeneity (‘g-strain’).

hemes in cytochromes apparently contributes to lowering of their reduction potentials [5-71. Given the -2 charge of the porphyrin, the net charge of the Fe(III)-porphyrin environment (ignoring charged porphyrin substituents) is +l, whereas the reduced Fe(II)-porphyrin has a net local charge of 0. In general, the more highly charged oxidation state should be more stabilized in an environment of higher polarity (higher solvent accessibility). In cytochromes, greater solvent exposure stabilizes the oxidized state relative to the reduced state, causing a lowering of the reduction potential. The effective net charge on the oxidized Rd Fe(III)(S--Cys)4site is -1, whereas the reduced Fe(II)(S--Cys)4 site displays an effective net charge of -2. Therefore, one expects the reduced Fe(Cys), site of Rd to be relatively stabilized in a higher polarity environment, so that increased solvent accessibility should cause an increase in the reduction potential. If the substitutions of Val-8 and Leu-41 by charged residues resulted in side-chain conformations that increased the solvent accessibility of the Fe(Cys)4site in both oxidation states, the increases observed in the reduction potentials could be qualitatively rationalized. Since charged side chains can be better solvated than the hydrocarbon side chains of Val or Leu, a relatively extended conformation for the substituted Arg and Asp side chains that increases exposure of the Fe(Cys), site to solvent might be expected. This hypothetical dominance of solvent accessibility is also consis-

tent with the insignificant change in the reduction potential observed for the [T5R,T7R] variant, since these residues are farther away from the Fe(Cys), site (Fig. 1, Table 2) and substitution is predicted to have little effect on solvent accessibility of this site. Also, given the large deletion in D. vulgaris rubrerythrin in a sequence region that constitutes the outer loop of the rubredoxin structure (in the bottom left of Fig. l), an increased solvent accessibility of the Fe(Cysksite in this domain of rubrerythrin might reasonably be predicted; this increase could explain the high reduction potential of this site. A very recent solvated molecular dynamics simulation of C. pasteurianum rubredoxin revealed a significantly increased flexibility in the Val-8 side chain in the reduced state, allowing a water molecule close approach to the Fe site [34]. This suggests that mutationally induced changes in solvent accessibility must be considered in both oxidation states. The general hypothesis that solvent accessibility is a significant factor in controlling Rd reduction potentials is difficult to assess in the absence of direct structural information on these variants. Calculation of the solvent accessible surface areas of the cysteine sulfurs in the energy-minimized wild-type crystal structure and variant model structures does not yield convincing evidence for increased solvent accessibility of the Fe(Cys),, site in these variants. An indirect approach to testing this hypothesis involves a new set of mutations substituting smaller or larger uncharged side chains for Val-8 and Leu41. Preliminary experiments suggest that the [V8A] variant exhibits the expected increase in reduction potential. Acknowledgements RAS acknowledges the invaluable mentorship and personal friendship of Harry B. Gray, to whom this work is dedicated in honor of his 60th birthday. This research was supported by the National Institutes of Health (GM50736). Instrumentation was partially supported by the NSF Research Training Group Award to the Center for Metalloenzyme Studies (DIR 90-14281). We thank Ms. Laura Popovich for help with the CV measurements and Mr. Hui Zhang for help with EPR measurements and initial computations. References [l]’ K.K. Rodgers and S.G. Sligar, J. Am. Gem. Sot., 113, (1991) 9419-9421. [2] B. Shen, D.R. Jollies, C.D. Stout, T.C. Diller, F.A. Armstrong, C.M.N. Gorst, G.N. La Mar, P.J. Stephens and B.K. Burgess, J. Biol. Chem., 269, (1994) 8564-8575. [3] G.R. Moore, FEBSZ&r., 161, (1983) 171-175. [4] D.C. Rees, J. Mol. BioL. 141, (1980) 323-326. [5] E. Stellwagen, Nature, 275, (1978) 73-74. [6] R.J. Kassner, Proc. Nad. Acad. Sci. USA, 69, (1972) 2263-2267. [7] G.J. Pielak, A.G. Mauk and M. Smith, Naiure, 313, (1985) 152154.

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