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• W2007201144 abstract "The [4Fe-4S]2+/+ cluster ofAzotobacter vinelandii ferredoxin I (FdI) has an unusually low reduction potential (E 0′) relative to other structurally similar ferredoxins. Previous attempts to raise thatE 0′ by modification of surface charged residues were unsuccessful. In this study mutants were designed to alter theE 0′ by substitution of polar residues for nonpolar residues near the cluster and by modification of backbone amides. Three FdI variants, P21G, I40N, and I40Q, were purified and characterized, and electrochemical E 0′measurements show that all had altered E 0′relative to native FdI. For P21G FdI and I40Q FdI, theE 0′ increased by +42 and +53 mV, respectively validating the importance of dipole orientation in control ofE 0′. Protein Dipole Langevin Dipole calculations based on models for those variants accurately predicted the direction of the change in E 0′ while overestimating the magnitude. For I40N FdI, initial calculations based on the model predicted a +168 mV change in E 0′while a −33 mV change was observed. The x-ray structure of that variant, which was determined to 2.8 Å, revealed a number of changes in backbone and side chain dipole orientation and in solvent accessibility, that were not predicted by the model and that were likely to influence E 0′. Subsequent Protein Dipole Langevin Dipole calculations (using the actual I40N x-ray structures) did quite accurately predict the observed change inE 0′. The [4Fe-4S]2+/+ cluster ofAzotobacter vinelandii ferredoxin I (FdI) has an unusually low reduction potential (E 0′) relative to other structurally similar ferredoxins. Previous attempts to raise thatE 0′ by modification of surface charged residues were unsuccessful. In this study mutants were designed to alter theE 0′ by substitution of polar residues for nonpolar residues near the cluster and by modification of backbone amides. Three FdI variants, P21G, I40N, and I40Q, were purified and characterized, and electrochemical E 0′measurements show that all had altered E 0′relative to native FdI. For P21G FdI and I40Q FdI, theE 0′ increased by +42 and +53 mV, respectively validating the importance of dipole orientation in control ofE 0′. Protein Dipole Langevin Dipole calculations based on models for those variants accurately predicted the direction of the change in E 0′ while overestimating the magnitude. For I40N FdI, initial calculations based on the model predicted a +168 mV change in E 0′while a −33 mV change was observed. The x-ray structure of that variant, which was determined to 2.8 Å, revealed a number of changes in backbone and side chain dipole orientation and in solvent accessibility, that were not predicted by the model and that were likely to influence E 0′. Subsequent Protein Dipole Langevin Dipole calculations (using the actual I40N x-ray structures) did quite accurately predict the observed change inE 0′. ferredoxin 4-morpholineethanesulfonic acid 3-[tris(hydroxymethyl)methyl]aminopropanesulfonic acid Protein Dipole Langevin Dipole Protein Data Bank ohm(s) Iron-sulfur ([Fe-S]) proteins contain clusters composed of iron and inorganic sulfide atoms ligated to the protein primarily by cysteine residues. They are ubiquitous, and have diverse functions ranging from electron transfer to regulation of gene expression (for recent reviews, see Refs. 1Beinert H. Holm R.H. Münck E. Science. 1997; 277: 653-659Crossref PubMed Scopus (1519) Google Scholar, 2Holm R.H. Kennepohl P. Solomon E.I. Chem. Rev. 1996; 96: 2239-2314Crossref PubMed Scopus (2360) Google Scholar, 3Johnson M.K. King R.B. Encyclopedia of Inorganic Chemistry. John Wiley & Sons, New York1994: 1896-1915Google Scholar, 4Cammack R. Adv. Inorg. Chem. 1992; 38: 281-322Crossref Scopus (294) Google Scholar, 5Howard J.B. Rees D.C. Adv. Protein Chem. 1991; 42: 199-280Crossref PubMed Google Scholar, 6Beinert H. FASEB J. 1990; 4: 2483-2491Crossref PubMed Scopus (191) Google Scholar, 7Lindahl P.A. Kovacs J.A. J. Cluster Sci. 1990; 1: 29-73Crossref Scopus (36) Google Scholar). In order to carry out these different functions, individual proteins can dramatically alter the reactivity of [Fe-S] clusters in a number of ways. For example, by adding or subtracting iron and sulfide atoms to vary the cluster type (1Beinert H. Holm R.H. Münck E. Science. 1997; 277: 653-659Crossref PubMed Scopus (1519) Google Scholar, 2Holm R.H. Kennepohl P. Solomon E.I. Chem. Rev. 1996; 96: 2239-2314Crossref PubMed Scopus (2360) Google Scholar, 3Johnson M.K. King R.B. Encyclopedia of Inorganic Chemistry. John Wiley & Sons, New York1994: 1896-1915Google Scholar, 4Cammack R. Adv. Inorg. Chem. 1992; 38: 281-322Crossref Scopus (294) Google Scholar, 5Howard J.B. Rees D.C. Adv. Protein Chem. 1991; 42: 199-280Crossref PubMed Google Scholar, 6Beinert H. FASEB J. 1990; 4: 2483-2491Crossref PubMed Scopus (191) Google Scholar, 7Lindahl P.A. Kovacs J.A. J. Cluster Sci. 1990; 1: 29-73Crossref Scopus (36) Google Scholar), by bridging a cluster between two subunits (8Howard J.B. Rees D.C. Chem. Rev. 1996; 96: 2965-2982Crossref PubMed Scopus (912) Google Scholar, 9Golbeck J.H. Bryant D.A. Curr. Top. Bioenerg. 1991; 16: 83-177Crossref Google Scholar), by introducing non-cysteine ligands (8Howard J.B. Rees D.C. Chem. Rev. 1996; 96: 2965-2982Crossref PubMed Scopus (912) Google Scholar, 10Beinert H. Kennedy M.C. Stout C.D. Chem. Rev. 1996; 96: 2335-2373Crossref PubMed Scopus (478) Google Scholar, 11Aono S. Bryant F.O. Adams M.W.W. J. Bacteriol. 1989; 171: 3433-3439Crossref PubMed Google Scholar, 12Busch J.L.H. Breton J.L. Bartlett B.M. Armstrong F.A. James R. Thomson A.J. Biochem. J. 1997; 323: 95-102Crossref PubMed Scopus (43) Google Scholar), by bridging an [Fe-S] cluster to another prosthetic group (e.g. Ref. 13Crane B.R. Siegel L.M. Gotzoff E.D. Science. 1995; 270: 59-67Crossref PubMed Scopus (272) Google Scholar), by grouping multiple clusters in a particular order as revealed by the recent hydrogenase structures (13Crane B.R. Siegel L.M. Gotzoff E.D. Science. 1995; 270: 59-67Crossref PubMed Scopus (272) Google Scholar, 14Volbeda A. Garcin E. Piras C. Hatchikian E.C. Frey M. Fontecilla-Camps J.C. Nature. 1995; 373: 580-587Crossref PubMed Scopus (1387) Google Scholar, 15Peters J.W. Lanzilotta W.N. Lemon B.J. Seefeldt L.C. Science. 1998; 282: 1853-1858Crossref PubMed Google Scholar, 16Higuchi Y. Yagi T. Yasuoka N. Structure. 1997; 5: 1671-1680Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar) or by adding other metals or organic groups as occurs in the [Mo-7Fe-9S-homocitrate] FeMo cofactor sites of nitrogenase (17Chan M.K. Kim J. Rees D.C. Science. 1993; 260: 792-794Crossref PubMed Scopus (489) Google Scholar). For this study it is especially important to note that, even without modification of [Fe-S] type and organization, proteins are still able to control the reactivities of the clusters they contain. For example, [4Fe-4S] clusters with four cysteine ligands can utilize three different redox couples. The +3/+2 couple is used in a class of proteins designated the high potential iron proteins (18Bertini I. Gori-Savellini G. Luchinat C. J. Bio. Inorg. Chem. 1997; 2: 114-118Crossref Scopus (50) Google Scholar, 19Przysiecki C.T. Meyer T.E. Cusanovich M.A. Biochemistry. 1985; 24: 2542-2549Crossref PubMed Scopus (68) Google Scholar, 20Luchinat C. Capozzi F. Borsari M. Battistuzzi G. Sola M. Biochem. Biophys. Res. Commun. 1994; 203: 436-442Crossref PubMed Scopus (36) Google Scholar), the +2/+ couple is used in most ferredoxins and redox active enzymes (21Stephens P.J. Jollie D.R. Warshel A. Chem. Rev. 1996; 96: 2491-2513Crossref PubMed Scopus (293) Google Scholar), and the +/0 couple has recently been reported for the iron protein (Fe protein) of nitrogenase (22Watt G.D. Reddy K.R.N. J. Inorg. Biochem. 1994; 53: 281-294Crossref Scopus (102) Google Scholar, 23Angove H.C. Yoo S.J. Burgess B.K. Münck E. J. Am. Chem. Soc. 1997; 119: 8730-8731Crossref Scopus (103) Google Scholar). Even when a particular [4Fe-4S] redox couple has been selected the reactivity of these proteins can be extended further by protein modulation of the reduction potential (E 0′) of a particular redox couple. Thus, high potential iron proteins have potentials ranging from 90 to 450 mV (18Bertini I. Gori-Savellini G. Luchinat C. J. Bio. Inorg. Chem. 1997; 2: 114-118Crossref Scopus (50) Google Scholar, 19Przysiecki C.T. Meyer T.E. Cusanovich M.A. Biochemistry. 1985; 24: 2542-2549Crossref PubMed Scopus (68) Google Scholar, 20Luchinat C. Capozzi F. Borsari M. Battistuzzi G. Sola M. Biochem. Biophys. Res. Commun. 1994; 203: 436-442Crossref PubMed Scopus (36) Google Scholar, 21Stephens P.J. Jollie D.R. Warshel A. Chem. Rev. 1996; 96: 2491-2513Crossref PubMed Scopus (293) Google Scholar, 24Heering H.A. Bulsink Y.B.M. Hagen W.R. Meyer T.E. Eur. J. Biochem. 1995; 232: 811-817Crossref PubMed Scopus (39) Google Scholar, 25Iwagami S.G. Creach A.L. Haynes C.A. Borsari M. Felli I.C. Piccioli M. Eltis L.D. Protein Sci. 1995; 4: 2562-2572Crossref PubMed Scopus (38) Google Scholar, 26Soriano A. Li D. Bian S. Agarwal A. Cowan J.C. Biochemistry. 1996; 35: 12479-12486Crossref PubMed Scopus (46) Google Scholar, 27Bian S. Hemann C.F. Hille R. Cowan J.A. Biochemistry. 1996; 35: 14544-14552Crossref PubMed Scopus (22) Google Scholar, 28Sola M. Cowan J.A. Gray H.B. Biochemistry. 1989; 28: 5261-5268Crossref PubMed Scopus (29) Google Scholar, 29Bertini I. Luchinat C. Rosato A. Prog. Biophys. Mol. Biol. 1996; 66: 43-80Crossref PubMed Scopus (65) Google Scholar), while ferredoxins that contain structurally indistinguishable [4Fe-4S]2+/+ clusters have reduction potentials ranging from −280 to −715 mV in different native proteins (21Stephens P.J. Jollie D.R. Warshel A. Chem. Rev. 1996; 96: 2491-2513Crossref PubMed Scopus (293) Google Scholar, 30Macedo A.L. Besson S. Moreno C. Fauque G. Moura J.J. Moura I. Biochem. Biophys. Res. Commun. 1996; 229: 524-530Crossref PubMed Scopus (12) Google Scholar). This study is focused on the question of how a protein could control the E 0′ of a [4Fe-4S]2+/+ cluster that is ligated via a typical CysXXCysXXCys motif and one remote Cys ligand. Early studies of protein control of [4Fe-4S]2+/+ E 0′ focused on three structurally characterized, related proteins. The [4Fe-4S]2+/+ cluster of Azotobacter vinelandiiferredoxin I (AvFdI)1 has an unusually low E 0′ of ∼ −630 mV at pH 8 (31Shen B. Martin L.L. Butt J.N. Armstrong F.A. Burgess B.K. J. Biol. Chem. 1993; 268: 25928-25939Abstract Full Text PDF PubMed Google Scholar), while the analogous clusters in Peptostreptococcus asaccharolyticus 2The organism P. asaccharolyticus was formerly named Peptococcus aerogenes. ferredoxin (PaFd) and Clostridium acidiuriciferredoxin (CaFd) have E 0′ ∼ −430 mV (32Stombaugh N.A. Sundquist J.E. Burris R.H. Orme-Johnson W.H. Biochemistry. 1976; 15: 2633-2841Crossref PubMed Scopus (110) Google Scholar). Thus the E 0′ for the [4Fe-4S]2+/+ clusters contained within these proteins vary by over 200 mV. Early comparisons of the structures and sequences for these three proteins showed that the peptide folding around the analogous clusters is highly conserved with respect to the location of the four Cys ligands, the Cys dihedral angles, and the eight amide groups H-bonded to sulfur atoms of the cluster (33Backes G. Mino Y. Loehr T.M. Meyer M.A. Cusanovich W.V. Sweeney W.V. Adman E.T. Sanders-Loehr J. J. Am. Chem. Soc. 1991; 113: 2055-2064Crossref Scopus (252) Google Scholar). These similarities have also been confirmed by the new 1.4-Å structures ofAvFdI (34Stout C.D. Stura E.A. McRee D.E. J. Mol. Biol. 1998; 278: 629-639Crossref PubMed Scopus (45) Google Scholar, 35Schipke C.G. Goodin D.B. McRee D.E. Stout C.D. Biochemistry. 1999; 38: 8228-8239Crossref PubMed Scopus (35) Google Scholar) and by the 0.95-Å structure ofCaFd (36Dauter Z. Wilson K.S. Sieker L.C. Meyer J. Moulis J.-M. Biochemistry. 1997; 36: 16065-16073Crossref PubMed Scopus (128) Google Scholar). Thus, these factors do not appear to be responsible for the observed differences in reduction potential among these proteins that all use the same [4Fe-4S]2+/+couple. Another long standing idea is that proteins might control [4Fe-4S]2+/+ E 0′ by introducing or removing charged residues. Thus, removing a negative surface charge near the cluster should make the cluster easier to reduce (raiseE 0′), while adding a negative charge should make it more difficult to reduce (lower E 0′) (18Bertini I. Gori-Savellini G. Luchinat C. J. Bio. Inorg. Chem. 1997; 2: 114-118Crossref Scopus (50) Google Scholar,37Rees D.C. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3082-3085Crossref PubMed Scopus (62) Google Scholar, 38Mauk A.G. Moore G.R. J. Biol. Inorg. Chem. 1997; 2: 119-125Crossref Scopus (108) Google Scholar, 39Naray-Szabo G. J. Biol. Inorg. Chem. 1997; 2: 135-138Crossref Scopus (28) Google Scholar, 40Warshel A. Papazyan A. Muegge I. J. Biol. Inorg. Chem. 1997; 2: 143-152Crossref Scopus (105) Google Scholar, 41Moore G.R. FEBS Lett. 1983; 161: 171-175Crossref PubMed Scopus (115) Google Scholar, 42Schejter A. Eaton W.A. Biochemistry. 1984; 23: 1081-1084Crossref Scopus (30) Google Scholar). Indeed, the lower potential AvFdI does have more negatively charged residues near its [4Fe-4S]2+/+ cluster than CaFd or PaFd (43Shen B.H. Jollie D.R. Stout C.D. Diller T.C. Armstrong F.A. Gorst C.M. LaMar G.N. Stephens P.J. Burgess B.K. J. Biol. Chem. 1994; 269: 8564-8575Abstract Full Text PDF PubMed Google Scholar). This idea was attractive because it predicted that the formation of salt bridges between redox partners, when they bind to each other, might serve to raise the potential of the electron acceptor while lowering the potential of the electron donor, thus facilitating electron transfer (37Rees D.C. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3082-3085Crossref PubMed Scopus (62) Google Scholar). To test this idea a number of site-directed variants of the lowerE 0′ AvFdI were constructed by changing the negatively charged surface residues near its [4Fe-4S]2+/+ cluster to their neutral or positively charged counterparts in the higher E 0′ PaFd (43Shen B.H. Jollie D.R. Stout C.D. Diller T.C. Armstrong F.A. Gorst C.M. LaMar G.N. Stephens P.J. Burgess B.K. J. Biol. Chem. 1994; 269: 8564-8575Abstract Full Text PDF PubMed Google Scholar). X-ray structures of the mutant proteins proved that the orientations of the residues were the same in the mutantAvFdIs as they were in native PaFd. Surprisingly, however, the E 0′ of the AvFd [4Fe-4S]2+/+ cluster was unaffected by these mutations (43Shen B.H. Jollie D.R. Stout C.D. Diller T.C. Armstrong F.A. Gorst C.M. LaMar G.N. Stephens P.J. Burgess B.K. J. Biol. Chem. 1994; 269: 8564-8575Abstract Full Text PDF PubMed Google Scholar). The conclusion from that study was that differences in surface charged residues were not responsible for the large differences in reduction potential observed for the [4Fe-4S]2+/+clusters of AvFdI and PaFd. Another factor that has been suggested to be important is the relative solvent accessibility of the [4Fe-4S]2+/+ clusters in the two classes of proteins with one group predicting that the higherE 0′ of PaFd arises from the presence of buried water molecules (21Stephens P.J. Jollie D.R. Warshel A. Chem. Rev. 1996; 96: 2491-2513Crossref PubMed Scopus (293) Google Scholar, 44Jensen G.M. Warshel A. Stephens P.J. Biochemistry. 1994; 33: 10911-10924Crossref PubMed Scopus (111) Google Scholar, 45Langen R. Jensen G.M. Jacob U. Stephens P.J. Warshel A. J. Biol. Chem. 1992; 267: 25625-25627Abstract Full Text PDF PubMed Google Scholar). A recent 0.95-Å resolution x-ray structure of a protein in the PaFd class, however, failed to reveal the presence of any internal water molecules (36Dauter Z. Wilson K.S. Sieker L.C. Meyer J. Moulis J.-M. Biochemistry. 1997; 36: 16065-16073Crossref PubMed Scopus (128) Google Scholar). Comparison of high resolution structures of AvFdI (34Stout C.D. Stura E.A. McRee D.E. J. Mol. Biol. 1998; 278: 629-639Crossref PubMed Scopus (45) Google Scholar, 35Schipke C.G. Goodin D.B. McRee D.E. Stout C.D. Biochemistry. 1999; 38: 8228-8239Crossref PubMed Scopus (35) Google Scholar) and CaFd (36Dauter Z. Wilson K.S. Sieker L.C. Meyer J. Moulis J.-M. Biochemistry. 1997; 36: 16065-16073Crossref PubMed Scopus (128) Google Scholar) also failed to reveal any significant differences in the solvent accessibility of the homologous [4Fe-4S]2+/+ clusters contained within the two proteins. In this study we use site-directed mutagenesis to manipulate side chain and backbone dipoles that are close to the [4Fe-4S]2+/+cluster of AvFdI (but not directly H-bonded to the sulfur atoms of the cluster) in order to examine an additional recent proposal that these factors are of critical importance in protein control ofE 0′ (21Stephens P.J. Jollie D.R. Warshel A. Chem. Rev. 1996; 96: 2491-2513Crossref PubMed Scopus (293) Google Scholar, 40Warshel A. Papazyan A. Muegge I. J. Biol. Inorg. Chem. 1997; 2: 143-152Crossref Scopus (105) Google Scholar, 44Jensen G.M. Warshel A. Stephens P.J. Biochemistry. 1994; 33: 10911-10924Crossref PubMed Scopus (111) Google Scholar, 45Langen R. Jensen G.M. Jacob U. Stephens P.J. Warshel A. J. Biol. Chem. 1992; 267: 25625-25627Abstract Full Text PDF PubMed Google Scholar). For mutagenesis T4 DNA ligase and T4 polynucleotide kinase were obtained from Life Technologies, Inc., while all restriction enzymes were from New England Biolabs (Beverly, MA). Thein vitro mutagenesis was performed as described previously (43Shen B.H. Jollie D.R. Stout C.D. Diller T.C. Armstrong F.A. Gorst C.M. LaMar G.N. Stephens P.J. Burgess B.K. J. Biol. Chem. 1994; 269: 8564-8575Abstract Full Text PDF PubMed Google Scholar) using a MutaGene M13 in vitro mutagenesis kit from Bio-Rad and the following oligonucleotides with the altered base(s) indicated in bold. The sequences are 5′-CCGGACGAGTGCCAGGACTGCGCGCTC-3′ for I40Q, 5′-CCGGACGAGTGCAACGACTGCGCGCTC-3′ for I40N, and 5′-GTTGAAGTCTGCGGCGTAGACTGTTTC-3′ for P21G. For all the mutants at Ile34 position, oligonucleotides with a mixed sequence were used to generate the mutants: 5′-GGGCCGAACTTCCTGGTC(CA)A(GC)CATCCGGACG-3′. The success of the mutagenesis was confirmed at the DNA level by dideoxy-DNA sequencing using the Sequenase version 2.0 DNA sequencing kit from Amersham Pharmacia Biotech. The overexpression of the FdI variants in their native background in A. vinelandii was carried out as described previously (46Vazquez A. Shen B. Negaard K. Iismaa S. Burgess B.K. Protein Exp. Purif. 1994; 5: 96-102Crossref PubMed Scopus (12) Google Scholar), except that the parent strain used for the overexpression was A. vinelandii LM100, a strain that does not synthesize native FdI, and electroporation (BTX TransPorator Plus™ electroporation system; BTX, Inc., San Diego, CA) was used instead of the triparental mating method in the transformation process. Cell growth and the purification and triclinic crystallization of native FdI and FdI variants was carried out as described previously (43Shen B.H. Jollie D.R. Stout C.D. Diller T.C. Armstrong F.A. Gorst C.M. LaMar G.N. Stephens P.J. Burgess B.K. J. Biol. Chem. 1994; 269: 8564-8575Abstract Full Text PDF PubMed Google Scholar, 47Stephens P.J. Jensen G.M. Devlin F.J. Morgan T.V. Stout C.D. Martı́n A.E. Burgess B.K. Biochemistry. 1991; 30: 3200-3209Crossref PubMed Scopus (47) Google Scholar). As a precautionary measure, the FdI variants were initially purified anaerobically in the presence of dithionite. The anaerobically purified protein was then exposed to the air to test its air stability. Once it was established that they were air-stable, further experiments including fast protein liquid chromatography (MonoQ, with a linear gradient of 0.15–0.5 m NaCl in Tris-HCl, pH 8.0) and triclinic crystallization (43Shen B.H. Jollie D.R. Stout C.D. Diller T.C. Armstrong F.A. Gorst C.M. LaMar G.N. Stephens P.J. Burgess B.K. J. Biol. Chem. 1994; 269: 8564-8575Abstract Full Text PDF PubMed Google Scholar) were done aerobically. For spectroscopic studies all samples were prepared anaerobically under argon in a Vacuum Atmospheres glove box (O2 < 1 ppm) using degassed buffers. Samples were first concentrated to the desired level using a Centricon-10 microconcentrator in 0.025 m Tris-HCl, pH 7.4. Reductions were carried out by addition of Na2S2O4 to 2.0 mm and incubation until no further change in absorption at >350 nm could be observed (usually this required about 20 min). UV-visible spectra were obtained with a Hewlett-Packard 8452 diode array UV-visible spectrophotometer. CD spectra were recorded using a Jasco J720 spectropolarimeter. EPR spectra were obtained using a Bruker 300 Ez spectrometer, interfaced with an Oxford Instruments ESR-9002 liquid helium continuous flow cryostat. TheE 0′ calculations were carried out using the program POLARIS, which was developed by Warshel et al. in the Department of Chemistry at the University of Southern California. This program is now commercially available from A. Warshel's group. In this study we used version 6.30 of this program on the spp2000 computer at the University of California, Irvine Office of Academic Computing. POLARIS was developed to calculate the free energies and electrostatic properties of molecules and macromolecules in solution using a Protein Dipole Langevin Dipole (PDLD) model. A detailed description of how the calculations are done for [Fe-S] proteins is found in Ref. 21Stephens P.J. Jollie D.R. Warshel A. Chem. Rev. 1996; 96: 2491-2513Crossref PubMed Scopus (293) Google Scholar and will only be briefly considered here. The calculations begin with a protein structure in a Protein Data Base (PDB) file format. In this case the starting point for calculations was the new 1.35-Å structure of the oxidized state of native FdI (34Stout C.D. Stura E.A. McRee D.E. J. Mol. Biol. 1998; 278: 629-639Crossref PubMed Scopus (45) Google Scholar) (7FDI). A number of PDB files were then created from that structure by modeling the I34N, I34Q, I40N, I40Q, or P21G mutations using the Insight II package (MSI, San Diego). The program replaces the wild-type residue with a mutant residue after local energy minimization. The actual I40N x-ray structures were also used as a starting point for calculations where indicated (accession code 1b0v). Once a structure had been obtained in PDB format, the next step was to convert that structure to a form suitable for PDLD calculations using a program called PREPARE that is included in the POLARIS package. This procedure involved deletion of all unwanted atoms (in this case the crystallographically observed ordered water molecules) and addition of H atoms if necessary. In the case of side chains that are capable of free rotation, the relative (Coulombic) energies of four orientations were evaluated and the configuration of minimum energy was selected (21Stephens P.J. Jollie D.R. Warshel A. Chem. Rev. 1996; 96: 2491-2513Crossref PubMed Scopus (293) Google Scholar). For His residues, the relative energies of the Nδd- and Nεe-protonated forms were evaluated similarly, and the configuration of minimum energy was chosen. Then, with the exceptions indicated below, all atoms were assigned charges including the atoms of the [Fe-S] cluster of interest, in this case the [4Fe-4S]Cys4 cluster in its oxidized and reduced states (21Stephens P.J. Jollie D.R. Warshel A. Chem. Rev. 1996; 96: 2491-2513Crossref PubMed Scopus (293) Google Scholar). The iron and inorganic sulfide atoms of the other [3Fe-4S]Cys3 cluster were treated as uncharged and assigned as zero. Additionally, the β-CH2and α-CH moieties of all ligating Cys residues were treated as uncharged, while non-ligand Cys residues were treated as normal amino acids. All the ionizable residues were treated as uncharged (total charge of the residue is zero). During the first part of the PDLD calculation, the Coulombic interactions of the [4Fe-4S]Cys4 cluster, in its oxidized and reduced states, with all other protein atoms was calculated. This included charge-charge interactions and charge-induced dipole interactions. The next part of the calculations involved construction of a Langevin dipole grid representing water molecules around the protein, and the interactions between the grid dipoles and the cluster were calculated. In this case the grid filled a sphere of radiusr L = 25 Å and was composed of two sections, an inner section with 1-Å spacing in a 12-Å radius and an outer section with a 3-Å spacing. For the oxidized state of the cluster, the dipoles on the constructed grid were optimized by sampling a set of 30 grids to give the maximum energy, and then the optimized grid was used without reoptimization for the reduced cluster. The final step involved calculation of the interaction of the cluster charges with the bulk media more than 25 Å away from the cluster. In the actual calculation process, the PDLD calculation is not only carried out on the starting protein structure, but also on molecular dynamics generated structures from the program. This is to take advantage of the fact that the average results based on a series of structures generated by molecular dynamics are more accurate than the single result from the x-ray crystal structure (21Stephens P.J. Jollie D.R. Warshel A. Chem. Rev. 1996; 96: 2491-2513Crossref PubMed Scopus (293) Google Scholar). The molecular dynamics simulations were done using a program called ENZYMIX that is attached to the POLARIS program. The molecular dynamics simulations were done at 300 K, generating a snapshot structure every 500 fs. A total of 50 structures were generated for each protein and each of them was subjected to PDLD calculation. The final results were averaged (21Stephens P.J. Jollie D.R. Warshel A. Chem. Rev. 1996; 96: 2491-2513Crossref PubMed Scopus (293) Google Scholar). Purified water of resistivity ∼18 MΩ cm (Millipore, Bedford, MA) was used in all experiments. The buffers MES, HEPES, and TAPS and the co-adsorbate neomycin sulfate were purchased from Sigma. An AutoLab electrochemical analyzer (EcoChemie, Utrecht, The Netherlands) was used to record DC voltammograms. The three-electrode configuration featuring all glass-cells has been described previously (48Armstrong F.A. Butt J.N. Sucheta A. Methods Enzymol. 1993; 227: 479-500Crossref PubMed Scopus (66) Google Scholar). The sample compartment (typically holding 500 μl) was maintained at 0 °C to optimize stability. AllE 0′ values are given with reference to the standard hydrogen electrode. The saturated calomel electrode was held at 22 °C which we have adopted as E (saturated calomel reference electrode) = +243 mV versus the standard hydrogen electrode. E 0′ values from cyclic voltammetry were calculated as the average of the anodic and the cathodic peak potentials, E 0′ = ½(E pa + E pc). The pyrolytic graphite “edge” electrode (surface area typically 0.18 cm2) was polished prior to each experiment with an aqueous alumina slurry (Buehler Micropolish; 1.0 μm) and then it was sonicated extensively to remove traces of Al2O3. All experiments were carried out under anaerobic conditions in a Vacuum Atmospheres glove box with an inert atmosphere of N2 (<1 ppm). Prior to the electrochemical experiments, all protein samples were checked for purity by running fast protein liquid chromatography (Amersham Pharmacia Biotech, Uppsala, Sweden) with a Mono Q column equilbrated with 0.05 m Tris-HCl, pH 7.4, and a linear gradient from 0 to 1 m NaCl. Bulk electrochemistry solutions contained 0.05–0.1 mm protein in 60 mm mixed buffer (15 mm HEPES, 15 mmMES, 15 mm TAPS, 15 mm acetate), with 0.1m NaCl as supporting electrolyte and 4 mmneomycin. Neomycin stabilizes the protein-electrode interactions. For the investigation of pH dependence, protein solutions were dialyzed extensively against the buffered solution at the required pH, using an Amicon 8MC diafiltration unit equipped with a microvolume assembly and a YM-3 membrane. Due to the altered solubility properties of the I40N, I40Q, and P21G variants of FdI with respect to the native protein, it was not possible to crystallize these mutants using the conditions for native FdI (49Stout C.D. J. Mol. Biol. 1989; 205: 545-555Crossref PubMed Scopus (137) Google Scholar) or seeding procedures (50Shen B. Jollie D.R. Diller T.C. Stout C.D. Stephens P.J. Burgess B.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10064-10068Crossref PubMed Scopus (48) Google Scholar). A large number of trials were carried out in an anaerobic glove box to screen for new crystallization conditions. For I40N FdI, brown, diamond-shaped, thin plates, approximately 0.3 × 0.3 × 0.05 mm in size, were grown by vapor diffusion from a solution containing 3 μl of 10 mg/ml protein in 0.4m Tris-HCl buffer, pH 7.8, and 3 μl of 4.2 m(NH4)2SO4 and 20 mmNaCl in 100 mm Tris-maleate buffer, pH 6.5 (reservoir solution). The crystallization droplets were equilibrated against 1.0 ml of the reservoir solution at room temperature, and crystals appeared after 4–5 days. Similar screening as well as seeding experiments with the I40Q and P21G mutants of FdI were unsuccessful. The thin, plate-like I40N FdI crystals exhibited highly mosaic diffraction in the direction parallel with the thin dimension of the crystal when frozen at 100 K using a variety of cryoprotectants. Therefore, data were collected from a single crystal of I40N FdI mounted aerobically in a thin-walled, glass capillary using the reservoir solution as a synthetic mother liquor. Data were collected at 18 °C using CuKα radiation from a Siemens SRA x-ray generator operated at 55 kV, 90 mA, and equipped with a graphite monochromator and a Mar Research 34.5-cm diameter image plate scanner. Data were recorded in 1° oscillations through a total rotation range of 162° with an exposure time of 10 min/frame. The data were indexed, integrated, merged, and scaled with Mosflm and Scala (51Leslie A.G.W. Acta Crystallogr. 1994; D50: 76" @default.
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