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.     

Structural origins of redox potentials in Fe-S proteins: electrostatic potentials of crystal structures.

Redox potentials often differ dramatically for homologous proteins that have identical redox centers. For two types of iron-sulfur proteins, the rubredoxins and the high-potential iron-sulfur proteins (HiPIPs), no structural explanations for these differences have been found. We calculated the classical electrostatic potential at the redox site using static crystal structures of four rubredoxins and four HiPIPs to identify important structural determinants of their redox potentials. The contributions from just the backbone and polar side chains are shown to explain major features of the experimental redox potentials. For instance, in the rubredoxins, the presence of Val 44 versus Ala 44 causes a backbone shift that explains a approximately 50 mV lower redox potential in one of the four rubredoxins. This result is consistent with experimental redox potentials of five additional rubredoxins with known sequence. Also, we attribute the unusually lower redox potentials of two of the HiPIPs studied to a less positive electrostatic environment around their redox sites. Finally, molecular dynamics simulations of solvent around static rubredoxin crystal structures indicate that water alone is a major factor in dampening the contribution of charged side chains, in accord with experiments showing that mutations of surface charges produce relatively little effect on redox potentials.     

Crystallographic studies of V44 mutants of Clostridium pasteurianum rubredoxin: effects of side-chain size on reduction potential

Understanding the structural origins of differences in reduction potentials is crucial to understanding how various electron transfer proteins modulate their reduction potentials and how they evolve for diverse functional roles. Here, the high-resolution structures of several Clostridium pasteurianum rubredoxin (Cp Rd) variants with changes in the vicinity of the redox site are reported in order to increase this understanding. Our crystal structures of [V44L] (at 1.8 A resolution), [V44A] (1.6 A), [V44G] (2.0 A) and [V44A, G45P] (1.5 A) Rd (all in their oxidized states) show that there is a gradual decrease in the distance between Fe and the amide nitrogen of residue 44 upon reduction in the size of the side chain of residue 44; the decrease occurs from leucine to valine, alanine or glycine and is accompanied by a gradual increase in their reduction potentials. Mutation of Cp Rd at position 44 also changes the hydrogen-bond distance between the amide nitrogen of residue 44 and the sulfur of cysteine 42 in a size-dependent manner. Our results suggest that residue 44 is an important determinant of Rd reduction potential in a manner dictated by side-chain size. Along with the electric dipole moment of the 43-44 peptide bond and the 44-42 NH--S type hydrogen bond, a modulation mechanism for solvent accessibility through residue 41 might regulate the redox reaction of the Rds.     

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.     

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.     

Protein control of electron transfer rates via polarization: molecular dynamics studies of rubredoxin

The protein matrix of an electron transfer protein creates an electrostatic environment for its redox site, which influences its electron transfer properties. Our studies of Fe-S proteins indicate that the protein is highly polarized around the redox site. Here, measures of deviations of the environmental electrostatic potential from a simple linear dielectric polarization response to the magnitude of the charge are proposed. In addition, a decomposition of the potential is proposed here to describe the apparent deviations from linearity, in which it is divided into a "permanent" component that is independent of the redox site charge and a dielectric component that linearly responds or polarizes to the charge. The nonlinearity measures and the decomposition were calculated for Clostridium pasteurianum rubredoxin from molecular dynamics simulations. The potential in rubredoxin is greater than expected from linear response theory, which implies it is a better electron acceptor than a redox site analog in a solvent with a dielectric constant equivalent to that of the protein. In addition, the potential in rubredoxin is described well by a permanent potential plus a linear response component. This permanent potential allows the protein matrix to create a favorable driving force with a low activation barrier for accepting electrons. The results here also suggest that the reduction potential of rubredoxin is determined mainly by the backbone and not the side chains, and that the redox site charge of rubredoxin may help to direct its folding.     

Amino acid sequence and function of rubredoxin from Desulfovibrio vulgaris Miyazaki

Rubredoxin was purified from Desulfovibrio vulgaris Miyazaki. It was sequenced and some of its properties determined. Rubredoxin is composed of 52 amino acids. It is highly homologous to that from D. vulgaris Hildenborough. Its N-terminal methionyl residue is partially formylated. The millimolar absorption coefficients of the rubredoxin at 489 nm and 280 nm are 8.1 and 18.5, respectively, and the standard redox potential is +5 mV, which is slightly higher than those of other rubredoxins. Rubredoxin, as well as cytochrome c-553, was reduced with lactate by the action of lactate dehydrogenase of this organism, and the reaction was stimulated with 2-methyl-1,4-naphthoquinone. It is suggested that rubredoxin, in collaboration with membranous quinone, functions as a natural electron carrier for cytoplasmic lactate dehydrogenase of this organism, whereas cytochrome c-553 plays the same role for periplasmic lactate dehydrogenase.     

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.     

Amino acid sequence and function of rebredoxin from Desulfovibrio vulgaris Miyazaki

Rubredoxin was purified from Desulfovibrio vulgaris Miyazaki. It was sequenced and some of its properties determined. Rubredoxin is composed of 52 amino acids. It is highly homologous to that from D. vulgaris Hildenborough. Its N-methionyl residue is partially formalated. The millimolar absorption coefficients of the rubredoxin at 489 nm and 280 are 8.1 and 18.5, respectively, and the standard redox potential is +5 mB, which is slightly higher than those of other rubredoxins. Rubredoxin, as well as cytochrome c-553, was reduced with lactate by the action of lactate dehydrogenase of this organism, and the rection was stimulated with 2-methyl-1, 4-naphthoquinone. It is suggested that rubredoxin, in collaboration with membraous quinone, functions as natural electron carrier for cytoplasmic lactate dehydrogenase of this organism, whereas cytochrome c-553 plays the same role for periplasmic lactate dehydrogenase.     

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.     

The unique hydrogen bonded water in the reduced form of<Emphasis Type="Italic"> Clostridium pasteurianum</Emphasis> rubredoxin and its possible role in electron transfer

Rubredoxin is a small iron-sulfur (FeS₄) protein involved in oxidation–reduction reactions. The side chain of Leu41 near the iron-sulfur center has two conformations, which we suggested previously serve as a gate for a water molecule during the electron transfer process. To establish the role of residue 41 in electron transfer, an [L41A] mutant of Clostridium pasteurianum rubredoxin was constructed and crystallized in both oxidation states. Despite the lack of the gating side chain in this protein, the structure of the reduced [L41A] rubredoxin reveals a specific water molecule in the same position as observed in the reduced wild-type rubredoxin. In contrast, both the wild-type and [L41A] rubredoxins in the oxidized state do not have water molecules in this location. The reduction potential of the [L41A] variant was ~50 mV more positive than wild-type. Based on these observations, it is proposed that the site around the S of Cys9 serves as a port for an electron acceptor. Lastly, the Fe–S distances of the reduced rubredoxin are expanded, while the hydrogen bonds between S of the cysteines and the backbone amide nitrogens are shortened compared to its oxidized counterpart. This small structural perturbation in the Fe(II)/Fe(III) transition is closely related to the small energy difference which is important in an effective electron transfer agent.     

Reduced temperature dependence of collective conformational opening in a hyperthermophile rubredoxin.

Spatially localized differences in the conformational dynamics of the rubredoxins from the hyperthermophile Pyrococcus furiosus (Pf) and the mesophile Clostridium pasteurianum (Cp) are monitored via amide exchange measurements. As shown previously for the hyperthermophile protein, nearly all backbone amides of the Cp rubredoxin exhibit EX(2) hydrogen exchange kinetics with conformational opening rates of >1 s(-)(1). Significantly slower amide exchange is observed for Pf rubredoxin in the region surrounding the metal site and the proximal end of the three-stranded beta-sheet, while for the rest of the structure, the exchange rates at 23 degrees C are similar for both proteins. For the multiple-turn region comprising residues 14-32 in both rubredoxins, the uniformity of both the exchange rate constants and the values of the activation energy at the slowly exchanging sites is consistent with a model of solvent exposure via a subglobal cooperative conformational opening. In contrast to the common expectation of increased rigidity in the hyperthermophile proteins, below room temperature Pf rubredoxin exhibits a larger apparent flexibility in this multiple-turn region. The smaller enthalpy for the conformational opening process of this region in Pf rubredoxin reflects the much weaker temperature dependence of the underlying conformational equilibrium in the hyperthermophile protein.     

The immunological properties of the Clostridium pasteurianum rubredoxin

An antibody to Clostridium pasteurianum rubredoxin was found in goat serum after multiple injections of the protein. This antibody was purified by adsorption and elution from a Sepharose-rubredoxin column. The purified antibody formed a precipitating complex with C. pasteurianum rubredoxin and aporubredoxin, but not with the rubredoxin from Micrococcus aerogenes, Peptostreptococcus elsdenii, and Pseudomonas oleovorans. All rubredoxins tested were adsorbed to Sepharose-antirubredoxin columns indicating that each could form a soluble complex with anti-C. pasteurianum rubredoxin. The relative affinity of the antirubredoxin for the various rubredoxins was demonstrated by its ability to inhibit the rubredoxin-dependent reduction of cytochrome c by NADPH in the presence of NADP-ferredoxin oxidoreductase. These data suggest that there are two antigenic determinants in C. pasteurianum rubredoxin and only one such determinant in the rubredoxin from other organisms which are recognized by anti-C. pasteurianum rubredoxin.     

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.     

Redox properties of mesophilic and hyperthermophilic rubredoxins as a function of pressure and temperature.

The formal equilibrium reduction potentials of recombinant electron transport protein, rubredoxin (MW = 7500 Da), from both the mesophilic Clostridium pasteurianum (Topt = 37 degrees C) and hyperthermophilic Pyrococcus furiosus (Topt = 95 degrees C) were recorded as a function of pressure and temperature. Measurements were made utilizing a specially designed stainless steel electrochemical cell that easily maintains pressures between 1 and 600 atm and a temperature-controlled cell that maintains temperatures between 4 and 100 degrees C. The reduction potential of P. furiosus rubredoxin was determined to be 31 mV at 25 degrees C and 1 atm, -93 mV at 95 degrees C and 1 atm, and 44 mV at 25 degrees C and 400 atm. Thus, the reduction potential of P. furiosus rubredoxin obtained under standard conditions is likely to be dramatically different from the reduction potential obtained under its normal operating conditions. Thermodynamic parameters associated with electron transfer were determined for both rubredoxins (for C. pasteurianum, DeltaV degrees = -27 mL/mol, DeltaS degrees = -36 cal K-1 mol-1, and DeltaH degrees = -10 kcal/mol, and for P. furiosus, DeltaV degrees = -31 mL/mol, DeltaS degrees = -41 cal K-1 mol-1, and DeltaH degrees = -13 kcal/mol) from its pressure- and temperature-reduction potential profiles. The thermodynamic parameters for electron transfer (DeltaV degrees, DeltaS degrees, and DeltaH degrees ) for both proteins were very similar, which is not surprising considering their structural similarities and sequence homology. Despite the fact that these two proteins exhibit dramatic differences in thermostability, it appears that structural changes that confer dramatic differences in thermostability do not significantly alter electron transfer reactivity. The experimental changes in reduction potential as a function of pressure and temperature were simulated using a continuum dielectric electrostatic model (DELPHI). A reasonable estimate of the protein dielectric constant (epsilonprotein) of 6 for both rubredoxins was determined from these simulations. A discussion is presented regarding the analysis of electrostatic interaction energies of biomolecules through pressure- and temperature-controlled electrochemical studies.     

Solution structure of a zinc substituted eukaryotic rubredoxin from the cryptomonad alga Guillardia theta

The rubredoxin from the cryptomonad Guillardia theta is one of the first examples of a rubredoxin encoded in a eukaryotic organism. The structure of a soluble zinc-substituted 70-residue G. theta rubredoxin lacking the membrane anchor and the thylakoid targeting sequence was determined by multidimensional heteronuclear NMR, representing the first three-dimensional (3D) structure of a eukaryotic rubredoxin. For the structure calculation a strategy was applied in which information about hydrogen bonds was directly inferred from a long-range HNCO experiment, and the dynamics of the protein was deduced from heteronuclear nuclear Overhauser effect data and exchange rates of the amide protons. The structure is well defined, exhibiting average root-mean-square deviations of 0.21 A for the backbone heavy atoms and 0.67 A for all heavy atoms of residues 7-56, and an increased flexibility toward the termini. The structure of this core fold is almost identical to that of prokaryotic rubredoxins. There are, however, significant differences with respect to the charge distribution at the protein surface, suggesting that G. theta rubredoxin exerts a different physiological function compared to the structurally characterized prokaryotic rubredoxins. The amino-terminal residues containing the putative signal peptidase recognition/cleavage site show an increased flexibility compared to the core fold, but still adopt a defined 3D orientation, which is mainly stabilized by nonlocal interactions to residues of the carboxy-terminal region. This orientation might reflect the structural elements and charge pattern necessary for correct signal peptidase recognition of the G. theta rubredoxin precursor.     

Eukaryotically encoded and chloroplast-located rubredoxin is associated with photosystem II

We analyzed a eukaryotically encoded rubredoxin from the cryptomonad Guillardia theta and identified additional domains at the N- and C-termini in comparison to known prokaryotic paralogous molecules. The cryptophytic N-terminal extension was shown to be a transit peptide for intracellular targeting of the protein to the plastid, whereas a C-terminal domain represents a membrane anchor. Rubredoxin was identified in all tested phototrophic eukaryotes. Presumably facilitated by its C-terminal extension, nucleomorph-encoded rubredoxin (nmRub) is associated with the thylakoid membrane. Association with photosystem II (PSII) was demonstrated by co-localization of nmRub and PSII membrane particles and PSII core complexes and confirmed by comparative electron paramagnetic resonance measurements. The midpoint potential of nmRub was determined as +125 mV, which is the highest redox potential of all known rubredoxins. Therefore, nmRub provides a striking example of the ability of the protein environment to tune the redox potentials of metal sites, allowing for evolutionary adaption in specific electron transport systems, as for example that coupled to the PSII pathway.     

Isolation and characterization of rubrerythrin, a non-heme iron protein from Desulfovibrio vulgaris that contains rubredoxin centers and a hemerythrin-like binuclear iron cluster

A new non-heme iron protein from the periplasmic fraction of Desulfovibrio vulgaris (Hildenbourough NCIB 8303) has been purified to homogeneity, and its amino acid composition, molecular weight, redox potential, iron content, and optical, EPR, and M?ssbauer spectroscopic properties have been determined. This new protein is composed of two identical subunits with subunit molecular weight of 21,900 and contains four iron atoms per molecule. The as-purified oxidized protein exhibits an optical spectrum with absorption maxima at 492, 365, and 280 nm, and its EPR spectrum shows resonances at g = 4.3 and 9.4, characteristic of oxidized rubredoxin. The M?ssbauer data indicate the presence of approximately equal amounts of two types of iron; we named them the Rd-like and the Hr-like iron due to their similarity to the iron centers of rubredoxins (Rds) and hemerythrins (Hrs), respectively. For the Rd-like iron, the measured fine and hyperfine parameters (D = 1.5 cm-1, E/D = 0.26, delta EQ = -0.55 mm/s, delta = 0.27 mm/s, Axx/gn beta n = -16.5 T, Ayy/gn beta n = -15.6 T, and Azz/gn beta n = -17.0 T) are almost identical with those obtained for the rubredoxin from Clostridium pasteurianum. Redox-titration studies monitored by EPR, however, showed that these Rd-like centers have a midpoint redox potential of +230 +/- 10 mV, approximately 250 mV more positive than those reported for rubredoxins. Another unusual feature of this protein is the presence of the Hr-like iron atoms.(ABSTRACT TRUNCATED AT 250 WORDS)