High Molecular Weight Cytochrome Research Articles

Role of the Iron Axial Ligands of Heme Carrier HasA in Heme Uptake and Release

The hemophore protein HasA from Serratia marcescens cycles between two states as follows: the heme-bound holoprotein, which functions as a carrier of the metal cofactor toward the membrane receptor HasR, and the heme-free apoprotein fishing for new porphyrin to be taken up after the heme has been delivered to HasR. Holo- and apo-forms differ for the conformation of the two loops L1 and L2, which provide the axial ligands of the iron through His(32) and Tyr(75), respectively. In the apo-form, loop L1 protrudes toward the solvent far away from loop L2; in the holoprotein, closing of the loops on the heme occurs upon establishment of the two axial coordination bonds. We have established that the two variants obtained via single point mutations of either axial ligand (namely H32A and Y75A) are both in the closed conformation. The presence of the heme and one out of two axial ligands is sufficient to establish a link between L1 and L2, thanks to the presence of coordinating solvent molecules. The latter are stabilized in the iron coordination environment by H-bond interactions with surrounding protein residues. The presence of such a water molecule in both variants is revealed here through a set of different spectroscopic techniques. Previous studies had shown that heme release and uptake processes occur via intermediate states characterized by a Tyr(75)-iron-bound form with open conformation of loop L1. Here, we demonstrate that these states do not naturally occur in the free protein but can only be driven by the interaction with the partner proteins.

Ferric iron reduction by Desulfovibrio vulgaris Hildenborough wild type and energy metabolism mutants

Desulfovibrio vulgaris Hildenborough wild type and its hyn1, hyd and hmc mutants, lacking genes for periplasmic [NiFe] hydrogenase-1, periplasmic [FeFe] hydrogenase or the transmembrane high molecular weight cytochrome (Hmc) complex, respectively, were able to reduce Fe(III) chelated with nitrilotriacetic acid (NTA), but not insoluble ferric oxide, with lactate as the electron donor. The rate and extent of Fe(III)-NTA reduction followed the order hyn = WT > hmc >> hyd, suggesting that reduction of soluble Fe(III) is a periplasmic process that requires the presence of periplasmic [FeFe] hydrogenase. Reduction of Fe(III)-NTA was not coupled to cell growth. In fact cell concentrations declined when D. vulgaris was incubated with Fe(III)-NTA as the only electron acceptor. Wild type and mutant cells reducing a limiting concentration of sulfate (2 mM), reduced Fe(III)-NTA with similar rates. However, these were similarly incapable of catalyzing subsequent lactate-dependent reduction of Fe(III)-NTA to completion. Periplasmic reduction of Fe(III)-NTA appeared to inhibit the productive, sulfate-reducing metabolism of D. vulgaris, possibly because it prevents the cycling of reducing equivalents needed to achieve a net bioenergetic benefit.

Interaction and electron transfer between the high molecular weight cytochrome and cytochrome c3 from Desulfovibrio vulgaris Hildenborough: Kinetic, microcalorimetric, EPR and electrochemical studies

The complex formation between the tetraheme cytochrome c3 and hexadecaheme high molecular weight cytochrome c (Hmc), the structure of which has recently been resolved, has been characterized by cross-linking experiments, EPR, electrochemistry and kinetic analysis, and some key parameters of the interaction were determined. The analysis of electron transfer between [Fe] hydrogenase, cytochrome c3 and Hmc demonstrates a redox-shuttling role of cytochrome c3 in the pathway from hydrogenase to Hmc, and shows an effect of redox state on the interaction between the two cytochromes. The role of polyheme cytochromes in electron transfer from periplasmic hydrogenase to membrane redox proteins is assessed. A model with cytochrome c3 as an intermediate between hydrogenase and various polyheme cytochromes is proposed and its physiological consequences are discussed.

Crystallization and MAD data collection of high-molecular weight cytochrome c from Desulfovibrio vulgaris Miyazaki F.

Hexadecaheme high molecular weight cytochrome c from a sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F has been successfully purified and crystallized. X-ray diffraction data have been collected by the multiple wavelength anomalous dispersion method. The crystal belongs to the space group P2(1)2(1)2(1) with unit-cell parameters a=60.42, b=84.29 and c=144.16 A and contains one molecule per asymmetric unit.

2P085 顕微分光で観測した高分子量チトクロム分子のX線照射による還元反応(ヘム蛋白質)

2P085 顕微分光で観測した高分子量チトクロム分子のX線照射による還元反応(ヘム蛋白質)

A novel multi-heme structure of high molecular-weight cytochrome C fromD. vulgaris Hildenborough

A novel multi-heme structure of high molecular-weight cytochrome C from<i>D. vulgaris Hildenborough</i>

2C1615 硫酸還元菌由来高分子量チトクロムcのX線結晶構造解析

2C1615 硫酸還元菌由来高分子量チトクロムcのX線結晶構造解析

The primary and three-dimensional structures of a nine-haem cytochrome c from Desulfovibrio desulfuricans ATCC 27774 reveal a new member of the Hmc family.

Haem-containing proteins are directly involved in electron transfer as well as in enzymatic functions. The nine-haem cytochrome c (9Hcc), previously described as having 12 haem groups, was isolated from cells of Desulfovibrio desulfuricans ATCC 27774, grown under both nitrate- and sulphate-respiring conditions. Models for the primary and three-dimensional structures of this cytochrome, containing 292 amino acid residues and nine haem groups, were derived using the multiple wavelength anomalous dispersion phasing method and refined using 1.8 A diffraction data to an R value of 17.0%. The nine haem groups are arranged into two tetrahaem clusters, with Fe-Fe distances and local protein fold similar to tetrahaem cytochromes c3, while the extra haem is located asymmetrically between the two clusters. This is the first known three-dimensional structure in which multiple copies of a tetrahaem cytochrome c3-like fold are present in the same polypeptide chain. Sequence homology was found between this cytochrome and the C-terminal region (residues 229-514) of the high molecular weight cytochrome c from Desulfovibrio vulgaris Hildenborough (DvH Hmc). A new haem arrangement in domains III and IV of DvH Hmc is proposed. Kinetic experiments showed that 9Hcc can be reduced by the [NiFe] hydrogenase from D. desulfuricans ATCC 27774, but that this reduction is faster in the presence of tetrahaem cytochrome c3. As Hmc has never been found in D. desulfuricans ATCC 27774, we propose that 9Hcc replaces it in this organism and is therefore probably involved in electron transfer across the membrane.

Association of cytochrome b5 with 16-androstene steroid synthesis in the testis and accumulation in the fat of male pigs.

The 16-androstene steroids, one of the principal causes of boar taint, are synthesized in the testis by the andien-beta synthase enzyme system. This system has been shown in vitro to involve both cytochrome P450c17 and cytochrome b5. The objective of this work was to investigate the relationship between the levels of cytochrome b5 in the testis, in vitro steroidogenesis, and the accumulation of 16-androstene steroids in the fat of pubertal boars. We found that the in vitro rate of 16-androstene steroidogenesis in testis microsomes was correlated with 16-androstene steroid concentrations in fat (r = .66, P < .01). Western blots were used to determine the amounts of cytochrome b5 and cytochrome P450c17 protein in testis, and two immunoreactive cytochrome b5 proteins of approximately 12 and 16 kDa were found. Levels of cytochrome P450c17 or the high molecular weight cytochrome b5 in testis were not significantly correlated to levels of 16-androstene steroids in fat. However, levels of total cytochrome b5 immunoreactive protein and levels of the low molecular weight immunoreactive cytochrome b5 were correlated to fat 16-androstene steroid concentrations (r = .59, P < .001; r = .72, P = .0001, respectively). Levels of the low molecular weight immunoreactive cytochrome b5 were also correlated to 16-androstene steroid synthesis rates in vitro (r = .62, P < .05). These results indicate that increased levels of a low molecular weight immunoreactive cytochrome b5 protein, and not of cytochrome P450c17, are related to increased testicular 16-androstene steroid production and accumulation in fat. These results support the hypothesis that selection for reduced levels of this low molecular weight immunoreactive cytochrome b5 protein in the testis may result in decreased levels of 16-androstene steroids in fat and reduced boar taint in uncastrated male pigs.

Characterization of the Cytochromes C fromDesulfovibrio desulfuricansG201

A monoheme cytochrome c553and a hexadecaheme high molecular weight cytochrome (Hmc) have been isolated and characterized from the sulfate-reducing bacteriaDesulfovibrio desulfuricansG201, in addition to the tetraheme cytochrome c3(Mr 13000) that has been previously described. Both cytochromes are homologous with respect to several biochemical properties to the corresponding cytochromes found in otherDesulfovibriospecies. However, they are acidic proteins while the corresponding molecules, isolated from otherDesulfovibriospecies, are relatively more basic. TheD. desulfuricanscytochrome content appears identical to that ofD. vulgarisHildenborough. Isolation of these cytochromes from aDesulfovibrio desulfuricansstrain is of great interest in order to get more insight on the physiological function of these molecules.

Benzoic acid 2-hydroxylase, a soluble oxygenase from tobacco, catalyzes salicylic acid biosynthesis.

Benzoic acid 2-hydroxylase (BA2H) catalyzes the biosynthesis of salicylic acid from benzoic acid. The enzyme has been partially purified and characterized as a soluble protein of 160 kDa. High-efficiency in vivo labeling of salicylic acid with 18O2 suggested that BA2H is an oxygenase that specifically hydroxylates the ortho position of benzoic acid. The enzyme was strongly induced by either tobacco mosaic virus inoculation or benzoic acid infiltration of tobacco leaves and it was inhibited by CO and other inhibitors of cytochrome P450 hydroxylases. The BA2H activity was immunodepleted by antibodies raised against SU2, a soluble cytochrome P450 from Streptomyces griseolus. The anti-SU2 antibodies immunoprecipitated a radiolabeled polypeptide of around 160 kDa from the soluble protein extracts of L-[35S]-methionine-fed tobacco leaves. Purified BA2H showed CO-difference spectra with a maximum at 457 nm. These data suggest that BA2H belongs to a novel class of soluble, high molecular weight cytochrome P450 enzymes.

Isolation and characterization of a high molecular weight cytochrome from the sulfate reducing bacterium Desulfovibrio gigas

A high molecular weight c-type cytochrome (Hmc) was purified and characterized from Desulfovibrio gigas. The molecular weight was estimated to be 67 kDa by SDS-PAGE and its N-terminus is homologous to those of the 16 hemes containing high molecular weight cytochrome c from Desulfovibrio vulgaris strains Hildenborough and Miyazaki. The purified hemoprotein shows c-type cytochrome absorption spectrum with ε 553(red) = 368 mM −1 · cm −1. A band at 640 nm, characteristic of high-spin hemes, was detected. The EPR spectra show the presence of two high-spin heme species, plus several non-equivalent low-spin hemes. The heme reduction potentials, at pH 7.6, range from −50 mV to −315 mV. In contrast to what has been described for D. vulgaris Hmc, the protein isolated from D. gigas directly accepts electrons from hydrogenase and further reduces other redox proteins.

Amino-acid sequence of the cytochrome c3 ( Mr 26 000) from Desulfovibrio desulfuricans Norway and a comparison with those of the other polyhemic cytochromes from Desulfovibrio

The amino-acid sequence of an octaheme cytochrome c 3 isolated from Desulfovibrio desulfuricans Norway is presented. The protein molecule ( M r 26 000) comprises two identical subunits of 111 amino acids with the characteristics typical of tetrahemic cytochrome c 3 class. Comparisons between the amino-acid sequences and physiological properties of cytochrome c 3 ( M r 26 000) and cytochromes c 3 ( M r 13 000) isolated from various species of Desulfovibrio showed the existence of considerable differences. In order to distinguish between the various subclasses in the cytochrome c 3 superfamily, the amino-acid sequence of cytochrome c 3 ( M r 26000) was compared with six known cytochrome c 3 ( M r 13000) sequences as well as with the sequence of the four c 3-like domains of a high molecular weight cytochrome c (Hmc) containing 16 hemes per molecule of 65 500 Da, isolated from Desulfovibrio vulgaris Hildenborough. The evolution and phylogenetic relationships of these various polyhemic cytochromes are discussed.

Biochemical and spectroscopic characterization of the high molecular weight cytochrome c from Desulfovibrio vulgaris Hildenborough expressed in Desulfovibrio desulfuricans G200.

The gene of high molecular weight, multiheme cytochrome c (Hmc) from the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough has been overexpressed in Desulfovibrio desulfuricans G200. The recombinant protein has been purified. Its molecular weight (65,600), amino acid composition, and NH2-terminal sequence were found to be identical to those of the wild-type protein. The recombinant protein has been spectroscopically characterized (optical spectrum, EPR, circular dichroism) and compared to the wild-type protein. We have found 16 hemes per molecule by iron analysis and the pyridine hemochrome test. Both high- and low-spin features were observed in the EPR spectrum. A detailed spin quantitation analysis indicates 1 or 2 high-spin hemes and 14 or 15 low-spin hemes per molecule. The redox potentials of the hemes determined by voltammetric techniques gave an average of three different values, 0, -100, and -250 mV (versus NHE), for the wild-type and the recombinant cytochrome. The low potential values are similar to the values observed for the bis(histidinyl) coordinated hemes of cytochrome c3. A comparison of the arrangement of heme binding sites and coordinated histidines in the amino acid sequences of cytochrome c3 and Hmc has shown that the latter contains four domains, three of which are complete c3-like domains, while the fourth represents an incomplete c3-like domain which may contain His-Met coordinated hemes. These data are in agreement with the detailed study of the number and types of hemes reported in this paper.

Isolation and crystallization of high molecular weight cytochrome from Desulfovibrio valgaris Hildenborough

High molecular weight cytochrome c from Desulfovibrio vulgaris Hildenborough has been isolated, purified and crystallized. The molecular weight was estimated to be 75 000 as the mean value from the results of gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein contains 16 c-type heme groups per molecule. The purified high molecular weight cytochrome c shows typical c-type cytochrome absorption spectra, with an α-peak at 553.2 nm ( ϵ 553.2 mM = 428) in the ferro form. The amino acid composition shows that the protein contains a sufficient number of cysteine and histidine residues to account for the high content of heme groups. The amino acid composition of D. vulgaris Hildenborough high molecular weight cytochrome c has no similarities to the D. desulfuricans hexahemoprotein nitrite reductase. The high content of the heme groups might suggest that the cytochrome has an alternate intrinsic biological function. Crystals of the high molecular weight cytochrome c have been grown from solutions of poly(ethylene glycol) 1000 or 2-methyl-2,4-pentanediol. The crystals are in space group P6 2 or P6 4 with unit cell dimensions a = b = 227.8, c = 105.7 Å and γ = 120°. A large unit cell volume of 4.75 · 10 6 Å 3 suggests that there are four or five protein molecules per asymmetric unit. They diffract to better than 4.0 Å resolution and appear to be resistant to radiation damage.