Protein control of iron-sulfur cluster redox potentials.

The relationship between the three-dimensional structures of iron-sulfur proteins and the redox potentials of their iron-sulfur clusters is of fundamental importance. We report calculations of the redox potentials of the [Fe4S4(S-cys)4]-2/-3 couple in four crystallographically characterized proteins: Azotobacter vinelandii ferredoxin I, Peptococcus aerogenes ferredoxin, Bacillus thermoproteolyticus ferredoxin, and Chromatium vinosum high potential iron protein (HiPIP). Our calculations use the "protein dipoles Langevin dipoles" microscopic electrostatic model, which includes both protein and solvent water. The variations in calculated redox potentials are in excellent agreement with experimental data. In particular, our results confirm the important role of amide groups close to the cluster in separating the potential of C. vinosum HiPIP from those of the other three proteins. However, the potentials of these latter exhibit a substantial range despite extremely similar amide group environments of their clusters. Our results show that the potentials in these proteins are tuned in part by varying the access of solvent water to the neighborhood of the cluster. Our calculations provide the first successful quantitative modeling of the protein control of iron-sulfur cluster redox potentials.     

The role of backbone stability near Ala44 in the high reduction potential class of rubredoxins

Rubredoxins may be separated into high and low reduction potential classes, with reduction potentials differing by approximately 50 mV. Our previous work showed that a local shift in the polar backbone due to an A(44) versus V(44) side-chain size causes this reduction potential difference. However, this work also indicated that in the low potential Clostridium pasteurianum (Cp) rubredoxin, a V(44) --> A(44) mutation causes larger local backbone flexibility, because the V(44) side-chain present in the wild-type (wt) is no longer present to interlock with neighboring residues to stabilize the subsequent G(45). Since Pyrococcus furiosus (Pf) and other high potential rubredoxins generally have a P(45), it was presumed that a G(45) --> P(45) mutation might stabilize a V(44) --> A(44) mutation in Cp rubredoxin. Here crystal structure analysis, energy minimization, and molecular dynamics (MD) were performed for wt V(44)G(45), single mutant A(44)G(45) and double mutant A(44)P(45) Cp, and for wt A(44)P(45) Pf rubredoxins. The local structural, dynamical, and electrostatic properties of Cp gradually approach wt Pf in the order wt Cp to single to double mutant because of greater sequence similarity, as expected. The double mutant A(44)P(45) Cp exhibits increased backbone stability near residue 44 and thus enhances the probability that the backbone dipoles point toward the redox site, which favors an increase in the electrostatic contribution to the reduction potential. It appears that the electrostatic potential of residue 44 and the solvent accessibility to the redox are both determinants for the reduction potentials of homologous rubredoxins. Overall, these results indicate that an A(44) in a rubredoxin may require a P(45) for backbone stability whereas a V(44) can accommodate a G(45), since the valine side-chain can interlock with its neighbors.     

Protein contributions to redox potentials of homologous rubredoxins: an energy minimization study.

The energetic contributions of the protein to the redox potential in an iron-sulfur protein are studied via energy minimization, comparing homologous rubredoxins from Clostridium pasteurianum, Desulfovibrio gigas, Desulfovibrio vulgaris, and Pyrococcus furiosus. The reduction reaction was divided into 1) the change in the redox site charge without allowing the protein to respond and 2) the relaxation of the protein in response to the new charge state, focusing on the latter. The energy minimizations predict structural relaxation near the redox site that agrees well with that in crystal structures of oxidized and reduced P. furiosus rubredoxin, but underpredicts it far from the redox site. However, the relaxation energies from the energy-minimized structures agree well with those from the crystal structures, because the polar groups near the redox site are the main determinants and the charged groups are all located at the surface and thus are screened dielectrically. Relaxation energies are necessary for good agreement with experimentally observed differences in reduction energies between C. pasteurianum and the other three rubredoxins. Overall, the relaxation energy is large (over 500 mV) from both the energy-minimized and the crystal structures. In addition, the range in the relaxation energy for the different rubredoxins is large (300 mV), because even though the structural perturbations of the polar groups are small, they are very near the redox site. Thus the relaxation energy is an important factor to consider in reduction energetics.     

Circular dichroism and redox properties of high redox potential ferredoxins

The circular dichroism (CD) spectra of 13 examples of high-potential iron-sulfur proteins (HiPIPs), a class of [4Fe-4S] ferredoxins, have been determined. In contrast to the proposal of Carter [Carter, C. W., Jr. (1977) J. Biol. Chem. 252, 7802-7811], no strict correlation between visible CD features and utilization of the [4Fe-4S]2+/[4Fe-4S]3+ oxidation levels was found. Although most HiPIPs have these features, the model requires their presence in all species. There is also no simple relationship between CD spectral features and the presence of conserved tyrosine-19. In addition, no apparent correlation between CD properties and oxidation-reduction potential could be detected. However, amino acid side chains in close contact to the iron-sulfur cluster appear to be important in modulating spectral and oxidation-reduction properties. In particular, the negative shoulder at 290 nm and negative maximum at 230 nm correlate with the presence of Trp-80 (Chromatium vinosum numbering). Two HiPIPs that do not have Trp at this position have positive bands at 290 and 230 nm. These bands in the Ectothiorhodospira halophila HiPIPs are apparently associated with Trp-49, which is located on the opposite side of the effective mirror plane of the cluster from Trp-80. The effect of pH on circular dichroism and redox potential in Thiocapsa roseopersicina HiPIP, which has a histidine at position 49, is consistent with the interaction of the side chain with the cluster. Despite specific differences in their CD spectra, the various HiPIPs studied show general similarity consistent with structural homology within this class of iron-sulfur proteins.     

Prediction of reduction potential changes in rubredoxin: a molecular mechanics approach

Predicting the effects of mutation on the reduction potential of proteins is crucial in understanding how reduction potentials are modulated by the protein environment. Previously, we proposed that an alanine vs. a valine at residue 44 leads to a 50-mV difference in reduction potential found in homologous rubredoxins because of a shift in the polar backbone relative to the iron site due to the different side-chain sizes. Here, the aim is to determine the effects of mutations to glycine, isoleucine, and leucine at residue 44 on the structure and reduction potential of rubredoxin, and if the effects are proportional to side-chain size. Crystal structure analysis, molecular mechanics simulations, and experimental reduction potentials of wild-type and mutant Clostridium pasteurianum rubredoxin, along with sequence analysis of homologous rubredoxins, indicate that the backbone position relative to the redox site as well as solvent penetration near the redox site are both structural determinants of the reduction potential, although not proportionally to side-chain size. Thus, protein interactions are too complex to be predicted by simple relationships, indicating the utility of molecular mechanics methods in understanding them.     

Fold versus sequence effects on the driving force for protein‐mediated electron transfer

Electron transport chains composed of electron transfer reactions mainly between proteins provide fast efficient flow of energy in a variety of metabolic pathways. Reduction potentials are essential characteristics of the proteins because they determine the driving forces for the electron transfers. As both polar and charged groups from the backbone and side chains define the electrostatic environment, both the fold and the sequence will contribute. However, although the role of a specific sequence may be determined by experimental mutagenesis studies of reduction potentials, understanding the role of the fold by experiment is much more difficult. Here, continuum electrostatics and density functional theory calculations are used to analyze reduction potentials in [4Fe‐4S] proteins. A key feature is that multiple homologous proteins in three different folds are compared: six high potential iron‐sulfur proteins, four bacterial ferredoxins, and four nitrogenase iron proteins. Calculated absolute reduction potentials are shown to be in quantitative agreement with electrochemical reduction potentials. Calculations further demonstrate that the contribution of the backbone is larger than that of the side chains and is consistent for homologous proteins but differs for nonhomologous proteins, indicating that the fold is the major protein factor determining the reduction potential, whereas the specific amino acid sequence tunes the reduction potential for a given fold. Moreover, the fold contribution is determined mainly by the proximity of the redox site to the protein surface and the orientation of the dipoles of backbone near the redox site. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Determining Rieske cluster reduction potentials

The Rieske iron-sulfur proteins have reduction potentials ranging from -150 to +400 mV. This enormous range of potentials was first proposed to be due to differing solvent exposure or even protein structure. However, the increasing number of available crystal structures for Rieske iron-sulfur proteins has shown this not to be the case. Colbert and colleagues proposed in 2000 that differences in the electrostatic environment, and not structural differences, of a Rieske proteins are responsible for the wide range of reduction potentials observed. Using computational simulation methods and the newly determined structure of Pseudomonas sp. NCIB 9816-4 naphthalene dioxygenase Rieske ferredoxin (NDO-F9816-4), we have developed a model to predict the reduction potential of Rieske proteins given only their crystal structure. The reduction potential of NDO-F9816-4, determined using a highly oriented pyrolytic graphite electrode, was -150+/-2 mV versus the standard hydrogen electrode. The predicted reduction potentials correlate well with experimentally determined potentials. Given this model, the effect of protein mutations can be evaluated. Our results suggest that the reduction potential of new proteins can be estimated with good confidence from 3D structures of proteins. The structure of NDO-F9816-4 is the most basic Rieske ferredoxin structure determined to date. Thus, the contributions of additional structural motifs and their effects on reduction potential can be compared with respect to this base structure.     

The Molecular Determinants of the Increased Reduction Potential of the Rubredoxin Domain of Rubrerythrin Relative to Rubredoxin

Based on the crystal structures, three possible sequence determinants have been suggested as the cause of a 285 mV increase in reduction potential of the rubredoxin domain of rubrerythrin over rubredoxin by modulating the polar environment around the redox site. Here, electrostatic calculations of crystal structures of rubredoxin and rubrerythrin and molecular dynamics simulations of rubredoxin wild-type and mutants are used to elucidate the contributions to the increased reduction potential. Asn¹⁶⁰ and His¹⁷⁹ in rubrerythrin versus valines in rubredoxins are predicted to be the major contributors, as the polar side chains contribute significantly to the electrostatic potential in the redox site region. The mutant simulations show both side chains rotating on a nanosecond timescale between two conformations with different electrostatic contributions. Reduction also causes a change in the reduction energy that is consistent with a linear response due to the interesting mechanism of shifting the relative populations of the two conformations. In addition to this, a simulation of a triple mutant indicates the side-chain rotations are approximately anticorrelated so whereas one is in the high potential conformation, the other is in the low potential conformation. However, Ala¹⁷⁶ in rubrerythrin versus a leucine in rubredoxin is not predicted to be a large contributor, because the solvent accessibility increases only slightly in mutant simulations and because it is buried in the interface of the rubrerythrin homodimer.     

Rationalization of the reduction potentials within the series of the high potential iron-sulfur proteins

An analysis of the factors affecting reduction potentials within a series of high potential iron-sulfur proteins (HiPIP) has been performed by calculating the different contributions to the variation of electrostatic energy upon addition of one electron to the oxidized form of the protein. Molecular dynamics calculations were used to generate model structures of HiPIPs for which X-ray data are not available, starting from the known structures of highly homologous proteins. We have calculated and analyzed the contributions to the electrostatic energy deriving from the net charges present on the surface of the protein, the partial charges present on the uncharged residues, the polarizability of the protein atoms, the solvent dipoles and polarizabilities. A positive correlation with the reduction potentials was found only for the contribution due to the net charge of the protein which, in the absence of other factors such as differences in the coordination properties and in reorganizational energy upon reduction, is proposed to represent the determining effect for the large variation in reduction potential within this series of biological electron carriers.     

Hydrogen-1 nuclear magnetic resonance investigation of high-potential iron-sulfur proteins from Ectothiorhodospira halophila and Ectothiorhodospira vacuolata: a comparative study of hyperfine-shifted resonances

Proton NMR spectra of the oxidized and reduced forms of high-potential iron-sulfur proteins (HiPIPs) were recorded at 200 MHz. The proteins studied were the HiPIPs I and II from Ectothiorhodospira halophila and Ectothiorhodospira vacuolata. Hyperfine-shifted peaks in spectra of the oxidized proteins were assigned to some of the protons of the cysteinyl ligands and aromatic residues at the active site on the basis of their chemical shifts, longitudinal relaxation times, and temperature-dependent behavior. The cysteinyl C beta-H protons were found to resonate downfield (about 100 ppm) and the C alpha-H protons upfield (about-25 ppm). This hyperfine shift pattern is consistent with the observed isotropic shift being contact in origin; it probably results from a pi-spin-transfer mechanism. The large magnitudes of the chemical shifts of peaks assigned to aromatic residues suggest that these residues interact with the iron-sulfur cluster via pi-pi overlap. Some of the hyperfine-shifted peaks observed in water were found to disappear in 2H2O solution. Such resonances probably arise from exchange-labile hydrogens of amino acid residues directly hydrogen bonded to the iron-sulfur cluster. In the case of HiPIPs I and II from E. vacuolata, whose spectra are similar except for the number of such peaks, the relative number of hydrogen bonds inferred to be present in the oxidized and reduced proteins qualitatively explains the difference between their midpoint redox potentials. On the other hand, for E. halophila HiPIPs I and II, consideration of the inferred number of hydrogen bonds alone fails to predict the sign of the difference between their midpoint redox potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence determination of reduction potentials by cysteinyl hydrogen bonds and peptide pipoles in [4Fe-4S] ferredoxins.

A sequence determinant of reduction potentials is reported for bacterial [4Fe-4S]-type ferredoxins. The residue that is four residues C-terminal to the fourth ligand of either cluster is generally an alanine or a cysteine. In five experimental ferredoxin structures, the cysteine has the same structural orientation relative to the nearest cluster, which is stabilized by the SH...S bond. Although such bonds are generally considered weak, indications that Fe-S redox site sulfurs are better hydrogen-bond acceptors than most sulfurs include the numerous amide NH...S bonds noted by Adman and our quantum mechanical calculations. Furthermore, electrostatic potential calculations of 11 experimental ferredoxin structures indicate that the extra cysteine decreases the reduction potential relative to an alanine by approximately 60 mV, in agreement with experimental mutational studies. Moreover, the decrease in potential is due to a shift in the polar backbone stabilized by the SH...S bond rather than to the slightly polar cysteinyl side chain. Thus, these cysteines can "tune" the reduction potential, which could optimize electron flow in an electron transport chain. More generally, hydrogen bonds involving sulfur can be important in protein structure/function, and mutations causing polar backbone shifts can alter electrostatics and thus affect redox properties or even enzymatic activity of a protein.     

Molecular dynamics simulations of rubredoxin from Clostridium pasteurianum: Changes in structure and electrostatic potential duringredox reactions

Molecular dynamics simulations of Clostridium pasteurianum rubredoxin in the oxidized and reduced forms have been performed. Good agreement between both forms and crystal data has been obtained (rms deviation of backbone atoms of 1.06 and 1.42 Å, respectively), which was due in part to the use of explicit solvent and counterions. The reduced form exhibits an unexpected structural change: the redox site becomes much more solvent-accessible, so that water enters a channel between the surface and the site, but with little actual structural rearrangement (the rms deviation of backbone atoms between the oxidized and reduced is 0.77 Å). The increase in solvent accessibility is also seen, although to a much lesser extent, between the oxidized and reduced crystal structures of Pyrococcus furiosus rubredoxin, but no high resolution crystal or nuclear magnetic resonance solution data exist for reduced C. pasteurianum rubredoxin. The electrostatic potential at the iron site and fluctuations in the potential, which contribute to both the redox and electron transfer properties, have also been evaluated for both the oxidized and the reduced simulations. These results show that the backbone plays a significant role (62–70 kcall/mol/e) and the polar sidechains contribute relatively little (0–4 kcal/mol/e) to the absolute electrostatic potential at the iron of rubredoxin for both forms. However, both groups contribute significantly to the change in redox state by becoming more polarized and more densely packed around the redox site upon reduction. Furthermore, these results show that the solvent becomes much more polarized in the reduced form than in the oxidized form, even excluding the penetrating water. Finally, the simulation indicates that the contribution of the charged side chains to the electrostatic potential is largely canceled by that of the counterions. © 1995 Wiley-Liss, Inc.     

Semi-continuum electrostatic calculations of redox potentials in photosystem I

The midpoint redox potentials (E(m)) of all cofactors in photosystem I from Synechococcus elongatus as well as of the iron-sulfur (Fe(4)S(4)) clusters in two soluble ferredoxins from Azotobacter vinelandii and Clostridium acidiurici were calculated within the framework of a semi-continuum dielectric approach. The widely used treatment of proteins as uniform media with single dielectric permittivity is oversimplified, particularly, because permanent charges are considered both as a source for intraprotein electric field and as a part of dielectric polarizability. Our approach overcomes this inconsistency by using two dielectric constants: optical epsilon(o)=2.5 for permanent charges pre-existing in crystal structure, and static epsilon(s) for newly formed charges. We also take into account a substantial dielectric heterogeneity of photosystem I revealed by photoelectric measurements and a liquid junction potential correction for E(m) values of relevant redox cofactors measured in aprotic solvents. We show that calculations based on a single permittivity have the discrepancy with experimental data larger than 0.7 V, whereas E(m) values calculated within our approach fall in the range of experimental estimates. The electrostatic analysis combined with quantum chemistry calculations shows that (i) the energy decrease upon chlorophyll dimerization is essential for the downhill mode of primary charge separation between the special pair P(700) and the primary acceptor A(0); (ii) the primary donor is apparently P(700) but not a pair of accessory chlorophylls; (iii) the electron transfer from the A branch quinone Q(A) to the iron-sulfur cluster F(X) is most probably downhill, whereas that from the B branch quinone Q(B) to F(X) is essentially downhill.     

Leucine 41 is a gate for water entry in the reduction of Clostridium pasteurianum rubredoxin

Biological electron transfer is an efficient process even though the distances between the redox moieties are often quite large. It is therefore of great interest to gain an understanding of the physical basis of the rates and driving forces of these reactions. The structural relaxation of the protein that occurs upon change in redox state gives rise to the reorganizational energy, which is important in the rates and the driving forces of the proteins involved. To determine the structural relaxation in a redox protein, we have developed methods to hold a redox protein in its final oxidation state during crystallization while maintaining the same pH and salt conditions of the crystallization of the protein in its initial oxidation state. Based on 1.5 A resolution crystal structures and molecular dynamics simulations of oxidized and reduced rubredoxins (Rd) from Clostridium pasteurianum (Cp), the structural rearrangements upon reduction suggest specific mechanisms by which electron transfer reactions of rubredoxin should be facilitated. First, expansion of the [Fe-S] cluster and concomitant contraction of the NH...S hydrogen bonds lead to greater electrostatic stabilization of the extra negative charge. Second, a gating mechanism caused by the conformational change of Leucine 41, a nonpolar side chain, allows transient penetration of water molecules, which greatly increases the polarity of the redox site environment and also provides a source of protons. Our method of producing crystals of Cp Rd from a reducing solution leads to a distribution of water molecules not observed in the crystal structure of the reduced Rd from Pyrococcus furiosus. How general this correlation is among redox proteins must be determined in future work. The combination of our high-resolution crystal structures and molecular dynamics simulations provides a molecular picture of the structural rearrangement that occurs upon reduction in Cp rubredoxin.     

Redox potentials of chlorophylls in the photosystem II reaction center

Water oxidation generating atmospheric oxygen occurs in photosystem II (PSII), a large protein-pigment complex located in the thylakoid membrane. The recent crystal structures at 3.2 and 3.5 A resolutions provide novel details on amino acid side chains, especially in the D1/D2 subunits. We calculated the redox potentials for one-electron oxidation of the chlorophyll a (Chla) molecules in PSII, considering the protein environment in atomic detail. The calculated redox potentials for the dimer Chla (P(D1/D2)) and accessory Chla (Chl(D1/D2)) were 1.11-1.30 V relative to the normal hydrogen electrode at pH 7, which is high enough for water oxidation. The D1/D2 proteins and their cofactors contribute approximately 390 mV to the enormous upshift of 470 mV compared to the redox potential of monomeric Chla in dimethylformamide. The other subunits are responsible for the remaining 80 mV. The high redox potentials of the two accessory Chla Chl(D1/D2) suggests that they also participate in the charge separation process.     

Structural alignment of ferredoxin and flavodoxin based on electrostatic potentials: implications for their interactions with photosystem I and ferredoxin-NADP reductase

The two proteins ferredoxin and flavodoxin can replace each other in the photosynthetic electron transfer chain of cyanobacteria and algae. However, structure, size, and composition of ferredoxin and flavodoxin are completely different. Ferredoxin is a small iron-sulfur protein (approximately 100 amino acids), whereas flavodoxin is a flavin-containing protein (approximately 170 amino acids). The crystal structure of both proteins from the cyanobacteria Anabeana PCC 7120 is known. We used these two protein structures to investigate the structural basis of their functional equivalence. We apply the Hodgkin index to quantify the similarity of their electrostatic potentials. The technique has been applied successfully in indirect drug design for the alignment of small molecule and bioisosterism elucidation. It requires no predefined atom-atom correspondences. As is known from experiments, electrostatic interactions are most important for the association of ferredoxin and flavodoxin with their reaction partners photosystem I and ferredoxin-NADP reductase. Therefore, use of electrostatic potentials for the structural alignment is well justified. Our extensive search of the alignment space reveals two alignments with a high degree of similarity in the electrostatic potential. In both alignments, ferredoxin overlaps completely with flavodoxin. The active sites of ferredoxin and flavodoxin rather than their centers of mass coincide in both alignments. This is in agreement with electron microscopy investigations on photosystem I cross-linked to ferredoxin or flavodoxin. We identify residues that may have the same function in both proteins and relate our results to previous experimental data.     

Criteria that discriminate between native proteins and incorrectly folded models

Various theoretical concepts, such as free energy potentials, electrostatic interaction potentials, atomic packing, solvent-exposed surface, and surface charge distribution, were tested for their ability to discriminate between native proteins and misfolded protein models. Misfolded models were constructed by introducing incorrect side chains onto polypeptide backbones: side chains of the alpha-helical hemerythrin were modeled on the beta-sheeted backbone of immunoglobulin VL domain, whereas those of the VL domain were similarly modeled on the hemerythrin backbone. CONGEN, a conformational space sampling program, was used to construct the side chains, in contrast to the previous work, where incorrect side chains were modeled in all trans conformations. Capability of the conformational search procedure to reproduce native conformations was gauged first by rebuilding (the correct) side chains in hemerythrin and the VL domain: constructs with r.m.s. differences from the x-ray side chains 2.2-2.4 A were produced, and many calculated conformations matched the native ones quite well. Incorrectly folded models were then constructed by the same conformational protocol applied to incorrect amino acid sequences. All CONGEN constructs, both correctly and incorrectly folded, were characterized by exceptionally small molecular surfaces and low potential energies. Surface charge density, atomic packing, and Coulomb formula-based electrostatic interactions of the misfolded structures and the correctly folded proteins were similar, and therefore of little interest for diagnosing incorrect folds. The following criteria clearly favored the native structures over the misfolded ones: 1) solvent-exposed side-chain nonpolar surface, 2) number of buried ionizable groups, and 3) empirical free energy functions that incorporate solvent effects.     

Crystallographic investigation of the role of aspartate 95 in the modulation of the redox potentials of Desulfovibrio vulgaris flavodoxin

The side chain of aspartate 95 in flavodoxin from Desulfovibrio vulgaris provides the closest negative charge to N(1) of the bound FMN in the protein. Site-directed mutagenesis was used to substitute alanine, asparagine, or glutamate for this amino acid to assess the effect of this charge on the semiquinone/hydroquinone redox potential (E(1)) of the FMN cofactor. The D95A mutation shifts the E(1) redox potential positively by 16 mV, while a negative shift of 23 mV occurs in the oxidized/semiquinone midpoint redox potential (E(2)). The crystal structures of the oxidized and semiquinone forms of this mutant are similar to the corresponding states of the wild-type protein. In contrast to the wild-type protein, a further change in structure occurs in the D95A mutant in the hydroquinone form. The side chain of Y98 flips into an energetically more favorable edge-to-face interaction with the bound FMN. Analysis of the structural changes in the D95A mutant, taking into account electrostatic interactions at the FMN binding site, suggests that the pi-pi electrostatic repulsions have only a minor contribution to the very low E(1) redox potential of the FMN cofactor when bound to apoflavodoxin. Substitution of D95 with glutamate causes only a slight perturbation of the two one-electron redox potentials of the FMN cofactor. The structure of the D95E mutant reveals a large movement of the 60-loop (residues 60-64) away from the flavin in the oxidized structure. Reduction of this mutant to the hydroquinone causes the conformation of the 60-loop to revert back to that occurring in the structures of the wild-type protein. The crystal structures of the D95E mutant imply that electrostatic repulsion between a carboxylate on the side chain at position 95 and the phenol ring of Y98 prevents rotation of the Y98 side chain to a more energetically favorable conformation as occurs in the D95A mutant. Replacement of D95 with asparagine has no effect on E(2) but causes E(1) to change by 45 mV. The D95N mutant failed to crystallize. The K(d) values of the protein FMN complex in all three oxidation-reduction states differ from those of the wild-type complexes. Molecular modeling showed that the conformational energy of the protein changes with the redox state, in qualitative agreement with the observed changes in K(d), and allowed the electrostatic interactions between the FMN and the surrounding groups on the protein to be quantified.     

Side-chain torsional potentials: effect of dipeptide, protein, and solvent environment.

Side-chain torsional potentials in the bovine pancreatic trypsin inhibitor are calculated from empirical energy functions by use of the known X-ray structure of the protein and the rigid-geometry mapping technique. The potentials are analyzed to determine the roles and relative importance of contributions from the dipeptide backbone, the protein, and the crystalline environment of solvent and other protein molecules. The structural characteristics of the side chains determine two major patterns of energy surfaces, E(X1,X2): a gamma-branched pattern and a pattern for longer, straight side chains (Arg, Lys, Glu, and Met). Most of the dipeptide potential curves and surfaces have a local minimum corresponding to the side-chain torsional angles in the X-ray structure. Addition of the protein forces sharpens and/or selects from these minima, providing very good agreement with the experimental conformation for most side chains at the surface or in the core of the protein. Inclusion of the crystalline environment produces still better results, especially for the side chains extending away from the protein. The results are discussed in terms of the details of the interactions due to the surrounding, calculated solvent-accessibility figures and the temperature factors derived from the crystallographic refinement of the pancreatic trypsin inhibitor.     

Redox potential of quinones in both electron transfer branches of photosystem I

The redox potentials of the two electron transfer (ET) active quinones in the central part of photosystem I (PSI) were determined by evaluating the electrostatic energies from the solution of the Poisson-Boltzmann equation based on the crystal structure. The calculated redox potentials are -531 mV for A1A and -686 mV for A1B. From these results we conclude the following. (i) Both branches are active with a much faster ET in the B-branch than in the A-branch. (ii) The measured lifetime of 200-290 ns of reduced quinones agrees with the estimate for the A-branch and corroborates with an uphill ET from this quinone to the iron-sulfur cluster as observed in recent kinetic measurements. (iii) The electron paramagnetic resonance spectroscopic data refer to the A-branch quinone where the corresponding ET is uphill in energy. The negative redox potential of A1 in PSI is primarily because of the influence from the negatively charged FX, in contrast to the positive shift on the quinone redox potential in bacterial reaction center and PSII that is attributed to the positively charged non-heme iron atom. The conserved residue Asp-B575 changes its protonation state after quinone reduction. The difference of 155 mV in the quinone redox potentials of the two branches were attributed to the conformation of the backbone with a large contribution from Ser-A692 and Ser-B672 and to the side chain of Asp-B575, whose protonation state couples differently with the formation of the quinone radicals.