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.     

Phylogenetic studies of two rubredoxins from sulfate reducing bacteria

Summary The sequences of two rubredoxins isolated from the sulfate reducing bacteria:Desulfovibrio vulgaris andDesulfovibrio gigas have been elucidated. They have similar sequences but many more differences occur than would be expected from two bacteria of the same genus. Of the 52 sites, only 37 are occupied by identical residues. The primary structures are compared with those of the anaerobic bacteria rubredoxins ofClostridium pasteurianum, Micrococcus aerogenes, Pseudomonas oleovorans andPeptostreptococcus elsdenii: only 12 identities are found, mostly in the two clusters that contain two iron-bound cysteines each. A phylogenetic tree based on the primary structures is presented and possible relations with plant and bacterial ferredoxins are discussed. A secondary and tertiary structure, stereochemically compatible with the sequence data, is proposed.To whom reprint requests should be addressed     

Glucose and Pyruvate Metabolism of Spirochaeta litoralis, an Anaerobic Marine Spirochete

The pathways of glucose and pyruvate metabolism in Spirochaeta litoralis, a free-living, strictly anaerobic marine spirochete, were studied. Addition of 0.2 to 0.4 M NaCl (final concentration) to suspending buffers prevented cell lysis and was necessary for gas evolution from various substrates by cell suspensions. The organism fermented glucose mainly to ethanol, acetate, CO(2), and H(2). Determination of radioactivity in products formed from (14)C-labeled glucose and assays of enzymatic activities in cell extracts indicated that S. litoralis catabolized glucose via the Embden-Meyerhof pathway. A clostridial-type clastic reaction was utilized by the spirochete to degrade pyruvate to acetyl-coenzyme A, CO(2), and H(2). Formation of acetate from acetyl-coenzyme A was catalyzed by phosphotransacetylase and acetate kinase. Nicotinamide adenine dinucleotide-dependent acetaldehyde and alcohol dehydrogenases converted acetyl-coenzyme A to ethanol. A reversible hydrogenase activity was detected in cell extracts. S. litoralis cell extracts contained a rubredoxin similar in spectral properties to other bacterial rubredoxins.     

Analysis of metal incorporation during overexpression of Clostridium pasteurianum rubredoxin by electrospray FTICR mass spectrometry

The rate of production of Clostridium pasteurianum rubredoxin overexpressed in Escherichia coli was examined by electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry. Previous work had shown that this heterologous expression resulted in isolation of both iron-containing (FeRd) and zinc-containing (ZnRd) rubredoxins. In the present work, minimally processed cell lysates of E. coli were analyzed in order to monitor the production of FeRd and ZnRd. The sensitivity of the measurement favored FeRd relative to ZnRd, and this differential sensitivity was quantitated using previously separated and purified rubredoxins. A time course study indicated that ZnRd and FeRd are produced simultaneously during overexpression, but at different rates. The ratio of the concentration of ZnRd to FeRd increased in a linear fashion during 3 h following induction of overexpression. Since only FeRds have been reported from native bacteria and archaea, the data suggest that either Zn2+ is sequestered from rubredoxins during native biosynthesis or that ZnRds may have escaped detection in the native microorganisms. ESI-FTICR mass spectrometry is shown to be a useful tool for monitoring metal insertion during protein biosynthesis.     

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.     

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.     

Early evolution of cellular electron transport: Molecular models for the ferredoxin-rubredoxin-flavodoxin region

Using information from conformation-predicting algorithms and X-ray data, a possible mode of structural evolution from an ancestral molecule of about 28 residues to bacterial ferredoxins and rubredoxins is suggested. The possibility of a further evolutionary pathway leading to flavodoxinlike proteins is also indicated.     

Rubredoxin from Desulfovibrio gigas. A molecular model of the oxidized form at 1.4 A resolution

The crystal structure of rubredoxin from the sulfate-reducing bacterium Desulfovibrio gigas has been determined at 1.4 A resolution (1 A = 0.1 nm) by X-ray diffraction methods; starting with a model of the isostructural rubredoxin from Desulfovibrio vulgaris. Refinement of the molecular model has been carried out by restrained least-squares techniques and Fourier series calculations. The present model includes a formyl at the N-terminal end and 121 possible sites for solvent molecules with full or partial occupancy, which corresponds to the modeling of nearly all the solvent medium. The crystallographic R factor against the data with 10 A greater than d greater than 1.4 A with F greater than 2 sig(F), is 0.136; and R = 0.140 when all the data are considered. The estimated average root-mean-square (r.m.s.) error on the positional parameters is about 0.12 A. The overall structural features of this molecule are close to those of the two highly refined rubredoxins from Clostridium pasteurianum and D. vulgaris. Superposition of these two molecules on the rubredoxin from D. gigas shows in both cases an overall r.m.s. deviation of 0.5 A for the atoms in the main-chain and of 0.4 A for the atoms in the side-chains that make up the hydrophobic core. The iron atom is co-ordinated to four cysteine sulfur atoms forming an almost regular tetrahedron, with Fe-SG distances ranging from 2.27 A to 2.31 A and angles varying from 103 degrees to 115 degrees. The intramolecular hydrogen-bonding pattern is quite comparable to those found in other proteins refined at high resolution. All the polar groups are involved in hydrogen bonds: intramolecular, intermolecular or with solvent molecules. The main structural differences from the other rubredoxins are in the nature and the distribution of some of the charged residues over the molecular surface. The possible influence of several structural factors on the intramolecular and intermolecular electron transfer properties such as the NH...SG bonds, the solvent exposure of the redox center, and the aromatic core is discussed. The conservation, during evolution, of a ring of acidic residues in the proximity of the FeSG4 center suggests that this ring may be implicated in the recognition processes between rubredoxins and their functional partners.     

NMR and X-ray analysis of structural additivity in metal binding site-swapped hybrids of rubredoxin

Background  

Chimeric hybrids derived from the rubredoxins of Pyrococcus furiosus (Pf) and Clostridium pasteurianum (Cp) provide a robust system for the characterization of protein conformational stability and dynamics in a differential mode. Interchange of the seven nonconserved residues of the metal binding site between the Pf and Cp rubredoxins yields a complementary pair of hybrids, for which the sum of the thermodynamic stabilities is equal to the sum for the parental proteins. Furthermore, the increase in amide hydrogen exchange rates for the hyperthermophile-derived metal binding site hybrid is faithfully mirrored by a corresponding decrease for the complementary hybrid that is derived from the less thermostable rubredoxin, indicating a degree of additivity in the conformational fluctuations that underlie these exchange reactions.     

Nucleotide sequence and organization of an H2-uptake gene cluster from Rhizobium leguminosarum bv. viciae containing a rubredoxin-like gene and four additional open reading frames.

The nucleotide sequence of a 3.2 kb region following the hydrogenase structural operon (hupSLCDEF) in the H2-uptake gene cluster from Rhizobium leguminosarum by viciae strain 128C53 has been determined. Five closely linked genes encoding products of 16.3 (HupG), 30.5 (HupH), 8.0 (HupI), 18.4 (HupJ) and 38.7 (HupK) kDa were identified 166 bp downstream from hupF. Transposon insertions into hupG, hupH, hupJ and hupK suppress the H2-oxidizing capability of the wild-type strain. The amino acid sequence deduced from hupI contains two Cys-X-X-Cys motifs, characteristic of rubredoxins, separated by 29 amino acid residues showing strong sequence homology with other bacterial rubredoxins. The amino acid-derived sequence from hupG and hupH showed homology to products from genes hyaE and hyaF of the operon encoding hydrogenase 1 from Escherichia coli, and hupJ and hupK were related to open reading frames identified in Rhodobacter capsulatus and Azotobacter vinelandii hydrogenase gene clusters. An involvement of the hupGHIJK gene cluster in redox reactions related to hydrogenase synthesis or activity is predicted on the basis of the function as electron carrier attributed to rubredoxin.     

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)     

Thermostabilization of proteins by diglycerol phosphate, a new compatible solute from the hyperthermophile Archaeoglobus fulgidus

Diglycerol phosphate accumulates under salt stress in the archaeon Archaeoglobus fulgidus (L. O. Martins, R. Huber, H. Huber, K. O. Stetter, M. S. da Costa, and H. Santos, Appl. Environ. Microbiol. 63:896-902, 1997). This solute was purified after extraction from the cell biomass. In addition, the optically active and the optically inactive (racemic) forms of the compound were synthesized, and the ability of the solute to act as a protecting agent against heating was tested on several proteins derived from mesophilic or hyperthermophilic sources. Diglycerol phosphate exerted a considerable stabilizing effect against heat inactivation of rabbit muscle lactate dehydrogenase, baker's yeast alcohol dehydrogenase, and Thermococcus litoralis glutamate dehydrogenase. Highly homologous and structurally well-characterized rubredoxins from Desulfovibrio gigas, Desulfovibrio desulfuricans (ATCC 27774), and Clostridium pasteurianum were also examined for their thermal stabilities in the presence or absence of diglycerol phosphate, glycerol, and inorganic phosphate. These proteins showed different intrinsic thermostabilities, with half-lives in the range of 30 to 100 min. Diglycerol phosphate exerted a strong protecting effect, with approximately a fourfold increase in the half-lives for the loss of the visible spectra of D. gigas and C. pasteurianum rubredoxins. In contrast, the stability of D. desulfuricans rubredoxin was not affected. These different behaviors are discussed in the light of the known structural features of rubredoxins. The data show that diglycerol phosphate is a potentially useful protein stabilizer in biotechnological applications.     

Thermostabilization of Proteins by Diglycerol Phosphate, a New Compatible Solute from the Hyperthermophile Archaeoglobus fulgidus

Diglycerol phosphate accumulates under salt stress in the archaeon Archaeoglobus fulgidus (L. O. Martins, R. Huber, H. Huber, K. O. Stetter, M. S. da Costa, and H. Santos, Appl. Environ. Microbiol. 63:896–902, 1997). This solute was purified after extraction from the cell biomass. In addition, the optically active and the optically inactive (racemic) forms of the compound were synthesized, and the ability of the solute to act as a protecting agent against heating was tested on several proteins derived from mesophilic or hyperthermophilic sources. Diglycerol phosphate exerted a considerable stabilizing effect against heat inactivation of rabbit muscle lactate dehydrogenase, baker's yeast alcohol dehydrogenase, and Thermococcus litoralis glutamate dehydrogenase. Highly homologous and structurally well-characterized rubredoxins from Desulfovibrio gigas, Desulfovibrio desulfuricans (ATCC 27774), and Clostridium pasteurianum were also examined for their thermal stabilities in the presence or absence of diglycerol phosphate, glycerol, and inorganic phosphate. These proteins showed different intrinsic thermostabilities, with half-lives in the range of 30 to 100 min. Diglycerol phosphate exerted a strong protecting effect, with approximately a fourfold increase in the half-lives for the loss of the visible spectra of D. gigas and C. pasteurianum rubredoxins. In contrast, the stability of D. desulfuricans rubredoxin was not affected. These different behaviors are discussed in the light of the known structural features of rubredoxins. The data show that diglycerol phosphate is a potentially useful protein stabilizer in biotechnological applications.     

Characterization of ferredoxin, flavodoxin, and rubredoxin from Clostridium formicoaceticum grown in media with high and low iron contents

Ferredoxin, flavodoxin, and rubredoxin were purified to homogeneity from Clostridium formicoaceticum and characterized. Variation of the iron concentration of the growth medium caused substantial changes in the concentrations of ferredoxin and flavodoxin but not of rubredoxin. The ferredoxin has a molecular weight of 6,000 and is a four iron-four sulfur protein with eight cysteine residues. The spectrum is similar to that of other ferredoxins. The molar extinction coefficients are 22.6 X 10(3) and 17.6 X 10(3) at 280 and 390 nm, respectively. From 100 g wet weight of cells grown with 3.6 microM iron and with 40 microM iron, 5 and 20 mg offerredoxin were isolated, respectively. The molecular weight of rubredoxin is 5,800 and it contains one iron and four cysteines. The UV-visible absorption spectrum is dissimilar to those of other rubredoxins in that the 373 nm absorption peak is quite symmetric, lacking the characteristic 350-nm shoulder found in other rubredoxins. The flavodoxin is a 14,500-molecular-weight protein which contains 1 mol of flavin mononucleotide per mol of protein. It forms a stable, blue semiquinone upon light irradiation in the presence of EDTA or during enzymatic reduction. When cells were grown in low-iron medium, flavodoxin constituted at least 2% of the soluble cell protein; however, it was not detected in extracts of cells grown in high-iron medium. The rubredoxin and ferredoxin expressed during growth in low-iron and high-iron media are identical as judged by iron, inorganic sulfide, and amino acid analysis, as well as light absorption spectroscopy.     

The complete amino acid sequence of rubredoxin from the green phototrophic bacterium Chlorobium thiosulphatophilum strain PM

A complete amino acid sequence for the rubredoxin from the photosynthetic bacterium Chlorobium thiosulphatophilum is proposed. The sequence, a single polypeptide chain of 53 amino acids, was deduced from the sequences of peptides obtained by chymotryptic, tryptic, thermolytic or mild acid digestion. The rubredoxin shows a high degree of sequence homology with rubredoxins from non-photosynthetic bacteria, and the evolutionary implications of this are considered.     

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.     

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.     

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.