Cytochromes c and P-450 as terminal acceptors in a reconstituted system of mitochondrial hydroxylation

Both cytochromes c and P-450 can be reduced in vitro by a mitochondrial hydroxylation chain consisting of flavoprotein, adrenodoxin and NADPH as electron donors. The effects of pH, ionic strength, content and stoichiometry of intermediate electron carriers on the reduction rate of cytochromes c and P-450 have been compared. The data obtained demonstrate that the reduction of these cytochromes proceeds by different ways. It has been found that cytochrome c has an inhibitory effect on cytochrome P-450 reduction. On the other hand, cytochrome P-450 remarkably activates the process of cytochrome c reduction.     

Cytochromes c of Nitrobacter winogradskyi and Thiobacillus novellus: structure, function and evolution.

The amino acid sequences of Thiobacillus novellus and Nitrobacter winogradskyi cytochromes c have been compared with those of cytochromes c from several other organisms. The two bacterial cytochromes resemble eukaryotic cytochromes c; 49 amino-acid residues are identical between T. novellus and horse cytochromes c, and 50 residues identical between N. winogradskyi and horse cytochromes c. However, their reactivity with cow cytochrome c oxidase is about 80% lower than the reactivity of eukaryotic cytochromes c with the cow mitochondrial oxidase, while they react with yeast cytochrome c peroxidase as rapidly as eukaryotic cytochromes c. The numbers of identical amino-acid residues between T. novellus and animal cytochromes c are 45-53 and those between N. winogradskyi and animal cytochromes c 47-53, while those between the two bacterial cytochromes and yeast and protozoan cytochromes c are around 40. Thus, N. winogradskyi and T. novellus cytochromes c are more similar to animal cytochromes c than to yeast and protozoan cytochromes c on the basis of the amino-acid sequence.     

Redox potentials of the photosynthetic bacterial cytochromes c2 and the structural bases for variability.

The cytochromes c2 of the Rhodospirillaceae show a much greater variation in redox potential and its pH dependence than the mitochondrial cytochromes c that have been studied. It is proposed that the range of redox potential for cytochromes c2 functioning as the immediate electron donor to photo-oxidised bacteriochlorophyll may be 345-395 mV at pH 5. Closely related cytochromes c2 with different redox potentials show patterns of amino acid substitution which are consistent with changes in hydrophobicity near the haem being at least a partial determinant of redox potential. More distantly related cytochromes are difficult to compare because of the large number of amino acid substitutions and the probability that there are subtle changes in overall peptide chain folding. The redox potential versus pH curves can be analysed in terms of either one ionisation in the oxidised form or two in the oxidised form and one in the reduced. The pK in the oxidised form at higher pH values can be correlated with the pK for the disappearance or shift of the near infrared absorption band located near 695 nm. The structural bases of these ionisations are not known but the possible involvement of the haem propionate residues is discussed.     

Characterization of cytochromes from Methanosarcina strain Göl and their involvement in electron transport during growth on methanol.

Methanosarcina strain G?1 was tested for the presence of cytochromes. Low-temperature spectroscopy, hemochrome derivative spectroscopy, and redox titration revealed the presence of two b-type (b559 and b564) and two c-type (c547 and c552) cytochromes in membranes from Methanosarcina strain G?1. The midpoint potentials determined were Em,7 = -135 +/- 5 and -240 +/- 11 mV (b-type cytochromes) and Em,7 = -140 +/- 10 and -230 +/- 10 mV (c-type cytochromes). The protoheme IX and the heme c contents were 0.21 to 0.24 and 0.09 to 0.28 mumol/g of membrane protein, respectively. No cytochromes were detectable in the cytoplasmic fraction. Of various electron donors and acceptors tested, only the reduced form of coenzyme F420 (coenzyme F420H2) and the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreonine phosphate (CoM-S-S-HTP) were capable of reducing and oxidizing the cytochromes at a high rate, respectively. Addition of CoM-S-S-HTP to reduced cytochromes and subsequent low-temperature spectroscopy revealed the oxidation of cytochrome b564. On the basis of these results, we suggest that one or several cytochromes participate in the coenzyme F420H2-dependent reduction of the heterodisulfide.     

Characterization of three soluble c-type cytochromes isolated from soybean root nodule bacteroids of Bradyrhizobium japonicum strain CC705

Three soluble, low molecular mass cytochromes c (Mr 8000-15,000) were isolated and purified from soybean root nodule bacteroids of Bradyrhizobium japonicum strain CC705. On the basis of their alpha: absorbance peaks in the reduced forms, they were named cytochromes c550, c552 and c555. Cytochrome c552 reacted very fast, c555 very slowly and c550 not at all with carbon monoxide. The complete amino acid sequence (73 residues) of cytochrome c552 was established which identifies it as a monoheme, class I cytochrome c with some remote similarity to the cytochrome c6 family.     

Senescent: a new Neurospora crassa nuclear gene mutant derived from nature exhibits mitochondrial abnormalities and a "death" phenotype

Fungi are capable of potentially unlimited growth. We resolved nuclear types from multinuclear mycelium of a phenotypically normal wild isolate of the fungus Neurospora intermedia by plating its uninucleate microconidia and obtained a strain which, unlike the "parent" strain, exhibited clonal senescence in subcultures. The mutant gene, senescent, was introgressed into N. crassa and mapped four map units to the right of the his-1 locus on linkage group VR. senescent is the first nuclear gene mutant of Neurospora derived from nature that shows the death phenotype. Death of the sen mutant occurred faster at 34 degrees C than at 22 or 26 degrees C. Measurements of oxygen uptake of conidia using respiratory inhibitors and the spectrophotometric analyses of mitochondrial cytochromes showed that in sen cultures grown at 34 degrees C, cytochromes b and aa(3) were present but cytochrome c was absent. By contrast at 26 degrees C, cytochromes b and c were present but cytochrome aa(3) was diminished in the late subcultures. This suggested that the sen mutation does not affect the potential to produce functional cytochromes. The deficiency of the respiratory chain cytochromes may not be the cause of death of the sen mutant because the cytochrome c and aa(3) mutants of N. crassa are capable of sustained growth whereas sen is not. Possible explanations for the observations are discussed.     

Stimulation of the respiratory chain of rat liver mitochondria between cytochrome c1 and cytochrome c by glucagon treatment of rats

Mitochondria from glucagon-treated rats oxidize succinate, but not ascorbate plus tetramethylphenylenediamine, faster in the uncoupled state than do control mitochondria. The rate of O(2) uptake in the presence of both substrates is equal to the sum of the rates of the O(2) uptake in the presence of either substrate alone. It is concluded that the mitochondrial respiratory chain is limited at some point between cytochromes b and c and that this step is regulated by glucagon. Measurement of the cytochrome spectra under uncoupled conditions in the presence of succinate and rotenone demonstrates a crossover between cytochromes c and c(1) when control mitochondria are compared with those from glucagon-treated rats, cytochrome c being more oxidized and cytochrome c(1) more reduced in control mitochondria. Under conditions where pyruvate metabolism is studied the control mitochondria are generally more oxidized than those from glucagon-treated rats, the redox state of cytochrome b-566 correlating with the rate of pyruvate metabolism in sucrose medium. However, when the redox state of the mitochondria is taken into account, a crossover between cytochromes c and c(1) is again apparent. The spectra of the b cytochromes are complex, but cytochrome b-562 appears to become more reduced relative to cytochrome b-566 in mitochondria from glucagon-treated rats than in control mitochondria. This can be explained by the existence of a more alkaline matrix in glucagon-treated rats, the redox potential for cytochrome b being pH-sensitive. It is concluded that glucagon stimulates electron flow between cytochromes c(1) and c. The physiological significance of these findings is discussed.     

An alternative to the accepted phylogeny of purple bacteria based on 16S rRNA: analyses of the amino acid sequences of cytochromes C2 and C556 from Rhodobacter (Rhodovulum) sulfidophilus

It is becoming increasingly apparent from complete genome sequences that 16S rRNA data, as currently interpreted, does not provide an unambiguous picture of bacterial phylogeny. In contrast, we have found that analysis of insertions and deletions in the amino acid sequences of cytochrome c2 has some advantages in establishing relationships and that this approach may have broad utility in acquiring a better understanding of bacterial relationships. The amino acid sequences of cytochromes c2 and c556 have been determined in whole or in part from four strains of Rhodobacter sulfidophilus. The cytochrome c2 contains three- and eight-residue insertions as well as a single-residue deletion in common with the large cytochromes c2 but in contrast to the small cytochromes c2 and mitochondrial cytochromes. In addition, the Rb. sulfidophilus protein shares a rare six- to seven-residue insertion with other Rhodobacter cytochromes c2. The cytochrome c556 is a low-spin class II cytochrome c homologous to the greater family of cytochromes c', which are usually high-spin. The similarity of cytochrome c556 to other species of class II cytochromes is consistent with the relationships deduced from comparisons of cytochromes c2. Thus, our results do not support placement of Rb. sulfidophilus in a separate genus, Rhodovulum, which was proposed primarily on the basis of 16S rRNA sequences. Instead, the Rhodobacter cytochromes c2 are distinct from those of other genera and species of purple bacteria and show a different pattern of relationships among species than reported for 16S rRNA.     

Structure of the soluble domain of cytochrome c(552) from Paracoccus denitrificans in the oxidized and reduced states

The crystal structure of the soluble domain of the membrane bound cytochrome c(552) (cytochrome c(552)') from Paracoccus denitrificans was determined using the multiwavelength anomalous diffraction technique and refined at 1.5 A resolution for the oxidized and at 1. 4 A for the reduced state. This is the first high-resolution crystal structure of a cytochrome c at low ionic strength in both redox states. The atomic model allowed for a detailed assessment of the structural properties including the secondary structure, the heme geometry and interactions, and the redox-coupled structural changes. In general, the structure has the same features as that of known eukaryotic cytochromes c. However, the surface properties are very different. Cytochrome c(552)' has a large strongly negatively charged surface part and a smaller positively charged area around the solvent-exposed heme atoms. One of the internal water molecules conserved in all structures of eukaryotic cytochromes c is also present in this bacterial cytochrome c. It contributes to the interactions between the side-chain of Arg36 and the heme propionate connected to pyrrole ring A. Reduction of the oxidized crystals does not influence the conformation of cytochrome c(552)' in contrast to eukaryotic cytochromes c. The oxidized cytochrome c(552)', especially the region of amino acid residues 40 to 56, appears to be more flexible than the reduced one.     

Electron-paramagnetic-resonance studies of structure and function of the two-haem enzymes Pseudomonas cytochrome c peroxidase and beef heart cytochrome c oxidase

Beef heart cytochrome c oxidase contains two cytochromes, a and a3, and Pseudomonas aeruginosa cytochrome c peroxidase has one high- and one low-potential c haem, cHP and cLP. The parallelism in co-ordination and spin states between cytochrome a and haem cHP on the one hand and between cytochrome a3 and haem cLP on the other is illustrated. The two latter haems become accessible to cyanide, when the former are reduced. Such reduction also leads to an activation of the enzymes. Mechanisms are presented in which ferryl forms of cytochromes a3 and haem cLP take part. The enzymes reach an oxidation state, formally the same as resting enzyme, but with different properties.     

Electrochemical analysis of microsomal cytochromes]

The electrochemical behaviour of the electron transfer proteins -- cytochromes b5, c and P-450 was studied by classical polarography and electrolysis with spectrophotometric monitoring. It was shown electrons are directly transferred from the electrode to oxidized cytochromes c and b5. Cytochrome P-450 is not reduced on the electrodes. However, the reduced inactivated from of cytochrome P-420 was detected at high potential values (-1,5 v).     

Cytochromes c550, c552, and c1 in the Electron Transport Network of Paracoccus denitrificans: Redundant or Subtly Different in Function?

Paracoccus denitrificans strains with mutations in the genes encoding the cytochrome c(550), c(552), or c(1) and in combinations of these genes were constructed, and their growth characteristics were determined. Each mutant was able to grow heterotrophically with succinate as the carbon and free-energy source, although their specific growth rates and maximum cell numbers fell variably behind those of the wild type. Maximum cell numbers and rates of growth were also reduced when these strains were grown with methylamine as the sole free-energy source, with the triple cytochrome c mutant failing to grow on this substrate. Under anaerobic conditions in the presence of nitrate, none of the mutant strains lacking the cytochrome bc(1) complex reduced nitrite, which is cytotoxic and accumulated in the medium. The cytochrome c(550)-deficient mutant did denitrify provided copper was present. The cytochrome c(552) mutation had no apparent effect on the denitrifying potential of the mutant cells. The studies show that the cytochromes c have multiple tasks in electron transfer. The cytochrome bc(1) complex is the electron acceptor of the Q-pool and of amicyanin. It is also the electron donor to cytochromes c(550) and c(552) and to the cbb(3)-type oxidase. Cytochrome c(552) is an electron acceptor both of the cytochrome bc(1) complex and of amicyanin, as well as a dedicated electron donor to the aa(3)-type oxidase. Cytochrome c(550) can accept electrons from the cytochrome bc(1) complex and from amicyanin, whereas it is also the electron donor to both cytochrome c oxidases and to at least the nitrite reductase during denitrification. Deletion of the c-type cytochromes also affected the concentrations of remaining cytochromes c, suggesting that the organism is plastic in that it adjusts its infrastructure in response to signals derived from changed electron transfer routes.     

Two types of cytochrome cd1 in the aerobic photosynthetic bacterium, Erythrobacter sp. OCh 114

Components I and II of cytochrome cd1 which had different spectral features were purified from the aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh 114. Component I showed an absorption maxima at 700 and 406 nm in the oxidized form, and at 621, 552.5, 548 and 416 nm in the reduced form. Component II showed an absorption maxima at 635 and 410 nm in the oxidized form and at 628, 552.5, 548 and 417 nm in the reduced form. The relative molecular mass, Mr, of both cytochromes was determined to be 135,000 with two identical subunits. Components I and II showed pI values of 7.6 and 6.8, respectively. The redox potential of hemes ranged from +234 mV to +242 mV, except for the heme d1 of component I (Em7 = +134 mV). Components I and II showed both cytochrome c oxidase and nitrite reductase activities. Cytochrome c oxidase activity was strongly inhibited by a low concentration of nitrite and cyanide. Erythrobacter cytochromes c-551 and c-552 were utilized as electron donors for the cytochrome c oxidase reaction. The high affinity of cytochrome c-552 to component II (Km = 1.27 microM) suggested a physiological significance for this cytochrome. Erythrobacter cytochromes cd1 are unique in their presence in cells grown under aerobic conditions as compared to other bacterial cytochromes cd1 which are formed only under denitrifying conditions.     

Stepwise reduction of cytochromes b-562, b-566 and b-558 in rat liver mitochondria.

1. Addition of KCN to aerobic, rotenone-inhibited rat liver mitochondria with out addition of substrate caused reduction of cytochromes b-562 (having an alpha-band at 562 nm at room temperature), c + c1, and a + a3. The effect of KCN on cytochrome b-562 was reversed by pentachlorophenol, though the effect of KCN on cytochromes c+c1 and a+a3 was not reversed by this uncoupler.2. Addition of ATP to aerobic, rat liver mitochondria inhibited with 500 muM KCN under conditions were cytochromes b-562, c+c1 and a+a3 were reduced, caused reduction of cytochrome b-566. The absorbance spectrum of cytochrome b-566 had an alpha-band at 565.5 nm, a beta-band at 538 nm and a gamma-band at 431 nm, but no shoulder around 558 nm at room temperature. 3. Addition of succinate to rotenone-KCN-inhibited and ATP-treated rat liver mitochondria under conditions where cytochromes b-566, b-562, c+c1 and a+a3 were already fully reduced, caused reduction of cytochrome b-558 (having an alpha-band at 558 nm, a beta-band at 527 nm and a gamma-band at 426 nm at room temperature) after exhaustion of molecular oxygen in the reaction medium, without any contribution from a long-wavelength species (cytochrome b-566). 4. It was concluded that the 558-nm band is not a short-wavelength shoulder of cytochrome b-566, but is due to a different species from cytochrome b-566.     

Electrophoretic and Immunological Differences Between the Cytochrome c3 of Desulfovibrio desulfuricans and that of Desulfovibrio vulgaris

The cytochrome c(3) of Desulfovibrio desulfuricans and that of D. vulgaris were purified to homogeneity as judged by disc gel electrophoresis and by ultracentrifugation. Both cytochromes had an oxidation-reduction potential of -205 +/- 5 mv at pH 7.0 and showed characteristic absorption bands at 525 and 553 nm in the reduced state. The molecular weights of the two cytochromes (calculated from sedimentation and diffusion data) were similar, with values of 13,500 to 14,300 for D. desulfuricans and 13,800 to 14,700 for D. vulgaris. The two cytochromes differed in their electrophoretic properties on Geon and polyacrylamide gel electrophoresis and did not share a common precipitating antigenic determinant as judged by immunodiffusion data.     

Characterization and amino acid sequences of cytochromes c6 from two strains of the green alga Chlorella vulgaris

Cytochromes c6 from the green algae Chlorella vulgaris CK-5 (CK5cyc6) and C. vulgaris CK-22 (CK22cyc6) were characterized and their amino acid sequences were analyzed. CK5cyc6 had a molecular mass of 9.3 kDa, isoelectric points of 3.0 (reduced) and 3.6 (oxidized), and a redox potential of +362 mV at pH 7.0. CK22cyc6 had a molecular mass of 9.5 kDa, isoelectric points of 2.9 (reduced) and 3.5 (oxidized), and a redox potential of +355 mV at pH 7.0. The absorption spectra of both cytochromes c6 showed 4 maxima in reduced form, and 2 maxima and a weak peak at 695 nm in oxidized form. The pyridine ferrohemochrome spectra indicated that their prosthetic group was heme c. These physicochemical properties were similar to those of other algal cytochromes c6. The amino acids (88 residues) of CK5cyc6 and CK22cyc6 were sequenced and the sequence motif -CXXCH-, which is typical of the heme-binding site of c-type cytochrome, was clearly confirmed in both cytochromes. Twenty-six amino acid residues were substituted, and the similarity score of each of them was 70.45%.     

Crystal structure of the oxidised and reduced acidic cytochrome c3from Desulfovibrio africanus.

Unique among sulphate-reducing bacteria, Desulfovibrio africanus has two periplasmic tetraheme cytochromes c3, one with an acidic isoelectric point which exhibits an unusually low reactivity towards hydrogenase, and another with a basic isoelectric point which shows the usual cytochrome c3reactivity. The crystal structure of the oxidised acidic cytochrome c3of Desulfovibrio africanus (Dva.a) was solved by the multiple anomalous diffraction (MAD) method and refined to 1.6 A resolution. Its structure clearly belongs to the same family as the other known cytochromes c3, but with weak parentage with those of the Desulfovibrio genus and slightly closer to the cytochromes c3of Desulfomicrobium norvegicum. In Dva.a, one edge of heme I is completely exposed to the solvent and surrounded by a negatively charged protein surface. Heme I thus seems to play an important role in electron exchange, in addition to heme III or heme IV which are the electron exchange ports in the other cytochromes c3. The function of Dva.a and the nature of its redox partners in the cell are thus very likely different.By alignment of the seven known 3D structures including Dva.a, it is shown that the structure which is most conserved in all cytochromes c3is the four-heme cluster itself. There is no conserved continuous protein structure which could explain the remarkable invariance of the four-heme cluster. On the contrary, the proximity of the heme edges is such that they interact directly by hydrophobic and van der Waals contacts. This direct interaction, which always involves a pyrrole CA-CB side-chain and its bound protein cysteine Sgammaatom, is probably the main origin of the four-heme cluster stability. The same kind of interaction is found in the chaining of the hemes in other multihemic redox proteins.The crystal structure of reduced Dva. a was solved at 1.9 A resolution. The comparison of the oxidised and reduced structures reveals changes in the positions of water molecules and polar residues which probably result from changes in the protonation state of amino acids and heme propionates. Water molecules are found closer to the hemes and to the iron atoms in the reduced than in the oxidised state. A global movement of a chain fragment in the vicinity of hemes III and IV is observed which result very likely from the electrostatic reorganization of the polypeptide chain induced by reduction.     

Isolation of water-soluble cytochromes from cyanobacteria by adsorption chromatography

A simple procedure using ammonium sulfate to fractionate water-soluble cytochromes c553 and c550 on Sephacryl S-200 gel is described. The usefulness of this procedure has been studied using the crude extracts of mesophilic cyanobacteria. It was found that almost all the cytochromes were adsorbed on to the gel at 2.34 M ammonium sulfate and were eluted at decreasing salt concentrations. The cytochromes were free of interfering phycobiliproteins and thus were suitable for the study of isoelectric points. It was also found that this procedure allowed a clear separation of the cytochromes based on their hydrophobicities. The order of elution was cytochrome c553, then cytochrome c550, indicating that c550 is more hydrophobic than c553. All these results show that this procedure provides both a simplified and an efficient purification of the cytochromes and insight into their surface properties. The cytochromes of Microcystis aeruginosa were purified to homogeneity using this procedure and other existing ones. Homogeneous cytochromes c553 and c550 were chromatographed on Sephacryl S-200 at 1.75 M (NH4)2SO4 and found to elute in the same order as reported earlier for the cytochromes in the crude extracts. In addition, cytochrome c550 was found to be more heat resistant and less water soluble than cytochrome c553.     

Succinate oxidation by coupled potato tuber mitochondria followed by rapid scan spectrometry

Oxidation of succinate by potato tuber mitochondria has been investigated from aerobiosis to complete anuerobiosis. Difference spectra of the various steps were recorded by a rapid scan spectrometer delivering averaged spectra every 3 s in the range 380 to 630 mm. The transitions between state 3 and 4 resulted in large redox changes, essentially for the b cytochromes, and in significant changes in the spectral baseline (light scattering). At anaerobiosis the cytochromes c, c₁ and a were reduced while cytochrome a, remained oxidized. – Addition of uncouplers in aerobiosis induced oxidation of the b cytochromes, and when anaerobiosis occurred cytochromes c, c₁a and a₃ were reduced simultaneously. When uncouplers were added in anaerobiosis a partial oxidation of the b cytochromes and the reduction of cytochrome a₃ were observed. These results are interpreted as the building up of a membrane potential, maximal in state 4 and stable after anaerobiosis. The cytochromes buried in the membrane equilibrate with the membrane potential, and their redox states are sensitive to the changes. Variations of membrane potential also induce changes in the light scattering by the mitochondrial membrane.     

Intergeneric and intrageneric conjugal transfer of plasmids pAMβ1, pIL205 and pIP501 in Lactobacillus sake

Abstract Three membrane-bound acid-stable cytochromes c with molecular masses of 46, 30 and 21 kDa were characterized from a new Thiobacillus ferrooxidans strain. They were solubilized with high concentrations of dodecylmaltoside at pH 8. The 30 kDa cytochrome c was purified to a homogeneous state as established by SDS-PAGE analysis. It showed an absorption peak at 410 nm in the oxidized form and at 418, 523 and 552 nm in the reduced form. The 46 kDa cytochrome c co-purified with a non-heme protein of 36 kDa. The amino acid composition and the N-terminal amino acid sequence of the 46 kDa cytochrome c were determined and compared with those of the soluble 14 kDa and the membrane-bound 21, 22.3 and 68 kDa cytochromes c isolated from two different strains. The results clearly show that this cytochrome is distinct from both the 22.3, 21 and 14 kDa cytochrome species, and exhibits some similarities with the 68 kDa cytochrome c as regards its amino acid composition.