Comparison of cytochromes from anaerobically and aerobically grown cells of Pseudomonas perfectomarinus.

Pseudomonas perfectomarinus (ATCC 14405) is a facultative anaerobe capable of either oxygen respiration or anaerobic nitrate respiration, i.e., denitrification. A comparative study of the electron transfer components of cells revealed five c-type cytochromes and cytochrome cd in the soluble fraction from anaerobically grown cells and four c-type cytochromes in the soluble fraction from aerobically grown cells. Purification procedures yielded three c-type cytochromes (designated c-551, c-554, and acidic c-type) from both kinds of cells as indicated by similarities in absorption spectra, molecular weight, and electrophoretic mobility. Cytochrome cd, a diheme c-type cytochrome (cytochrome c-552), and a split-alpha c-type cytochrome were recovered only from anaerobically grown cells. A c-type cytochrome with a low ratio of alpha to beta absorption peak heights was uniquely present in the aerobically grown cells. Liquid N2 temperature absorption spectroscopy on the membrane fraction from anaerobically grown cells revealed residual cytochrome cd as well as differences in the relative amounts of c-type and b-type cytochromes in membranes prepared from cells grown under the two different conditions.     

Presence of methyl sterol and bacteriohopanepolyol in an outer-membrane preparation from Methylococcus capsulatus (Bath)

Cytoplasmic/intracytoplasmic and outer membrane preparations of Methylococcus capsulatus (Bath) were isolated by sucrose density gradient centrifugation of a total membrane fraction prepared by disruption using a French pressure cell. The cytoplasmic and/or intracytoplasmic membrane fraction consisted of two distinct bands, Ia and Ib (buoyant densities 1.16 and 1.8 g ml-1, respectively) that together contained 57% of the protein, 68% of the phospholipid, 73% of the ubiquinone and 89% of the CN-sensitive NADH oxidase activity. The only apparent difference between these two cytoplasmic bands was a much higher phospholipid content for Ia. The outer membrane fraction (buoyant density 1.23 - 1.24 g ml-1) contained 60% of the lipopolysaccharide-associated, beta-hydroxypalmitic acid, 74% of the methylsterol, and 66% of the bacteriohopanepolyol (BHP); phospholipid to methyl sterol or BHP ratios were 6:1. Methanol dehydrogenase activity and a c-type cytochrome were also present in this outer membrane fraction. Phospholipase A activity was present in both the cytoplasmic membrane and outer membrane fractions. The unique distribution of cyclic triterpenes may reflect a specific role in conferring outer membrane stability in this methanotrophic bacterium.     

A partial resolution and reconstitution of the ammonia-oxidizing system of Nitrosomonas europaea: role of cytochrome c554

The cell-free ammonia-oxidizing system of Nitrosomonas europaea was resolved into three major fractions: a membrane fraction containing cytochrome a1 and c-type cytochromes, a fraction with hydroxylamine-cytochrome c reductase and a cytochrome c fraction. The ammonia-oxidizing activity was reconstituted by the combination of these three fractions. The activity was more consistently reconstituted by adding Nitrosomonas cytochrome c554 to the membrane fraction. The hydroxylamine-cytochrome c reductase activity of the membrane fraction increased with the addition of cytochrome c554, but the oxidation of hydroxylamine to nitrite required a further addition of cytochrome c552. The ammonia oxidation by the membrane plus cytochrome c554 was affected by the concentration of phosphate and the addition of bovine serum albumin, spermine, or MgCl2.     

Computational structure prediction provides a plausible mechanism for electron transfer by the outer membrane protein Cyc2 from Acidithiobacillus ferrooxidans

Cyc2 is the key protein in the outer membrane of Acidithiobacillus ferrooxidans that mediates electron transfer between extracellular inorganic iron and the intracellular central metabolism. This cytochrome c is specific for iron and interacts with periplasmic proteins to complete a reversible electron transport chain. A structure of Cyc2 has not yet been characterized experimentally. Here we describe a structural model of Cyc2, and associated proteins, to highlight a plausible mechanism for the ferrous iron electron transfer chain. A comparative modeling protocol specific for trans membrane beta barrel (TMBB) proteins in acidophilic conditions (pH ~ 2) was applied to the primary sequence of Cyc2. The proposed structure has three main regimes: Extracellular loops exposed to low‐pH conditions, a TMBB, and an N‐terminal cytochrome‐like region within the periplasmic space. The Cyc2 model was further refined by identifying likely iron and heme docking sites. This represents the first computational model of Cyc2 that accounts for the membrane microenvironment and the acidity in the extracellular matrix. This approach can be used to model other TMBBs which can be critical for chemolithotrophic microbial growth.     

Membranes of Rhodospirillum rubrum: isolation and physicochemical properties of membranes from aerobically grown cells.

Highly purified preparations of cytoplasmic and outer membrane were isolated from aerobically grown Rhodospirillum rubrum lysed by sequential treatment with lysozyme, ethylenediaminetetraacetate, and Brij 58. The membranes were resolved and separated from other cellular constitutents by a combination of velocity and isopyknic sedimentation in sucrose density gradients. On the basis of their appearance in electron micrographs and their protein profiles in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, these preparations appear to be quite similar to those obtained from other gram-negative bacteria. The cytoplasmic membrane fraction contained the majority of the total membrane-bound succinic dehydrogenase activity and was 10-fold enriched in b- and c-type cytochrome with respect to the outer membrane. The latter fraction was characterized by a much greater carbohydrate content and the presence of arachidic acid, which is typical of R. rubrum lipopolysaccharide. Their protein fatty acid, and overall chemical compositions suggested that these preparations were freer from cross-contamination than those obtained from R. rubrum with currently available methods.     

Preliminary characterization studies on the Neisseria catarrhalis respiratory electron transport chain

The Neisseria catarrhalis respiratory electron transport system was examined in a sonic type particulate membrane fraction and shown to have a moderately active succinate as well as nonpyridine nucleotide-dependent dl-lactate oxidoreductase and a very active tetramethyl-p-phenylenediamine oxidase. l-Malate and l-glutamate oxidation were found to be dependent on pyridine nucleotides and exclusively associated with a soluble (or nonmembranous) fraction. The primary cytochrome components in the electron transport system appear to be c-type in nature (555 nm and 550 nm) as well as cytochrome a(1) (600 nm) and cytochrome o.     

Cytochrome c biogenesis in mitochondria--Systems III and V

In c-type cytochromes, heme becomes covalently attached to the polypeptide chain by a reaction between the vinyl groups of the heme and cysteine thiols from the protein. There are two such cytochromes in mitochondria: cytochrome c and cytochrome c(1). The heme attachment is a post-translational modification that is catalysed by different biogenesis proteins in different organisms. Three types of biogenesis system are found or predicted in mitochondria: System I (the cytochrome c maturation system); System III (termed holocytochrome c synthase (HCCS) or heme lyase); and System V. This review focuses primarily on cytochrome c maturation in mitochondria containing HCCS (System III). It describes what is known about the enzymology and substrate specificity of HCCS; the role of HCCS in human disease; import of HCCS into mitochondria; import of apocytochromes c and c(1) into mitochondria and the close relationships with HCCS-dependent heme attachment; and the role of the fungal cytochrome c biogenesis accessory protein Cyc2. System V is also discussed; this is the postulated mitochondrial cytochrome c biogenesis system of trypanosomes and related organisms. No cytochrome c biogenesis proteins have been identified in the genomes of these organisms whose c-type cytochromes also have a unique mode of heme attachment.     

Membranes of Rhodopseudomonas sphaeroides. VII. Photochemical properties of a fraction enriched in newly synthesized bacteriochlorophyll a-protein complexes.

Previous pulse-chase studies have shown that bacteriochlorophyll a-protein complexes destined eventually for the photosynthetic (chromatophore) membrane of Rhodopseudomonas sphaeroides appear first in a distinct pigmented fraction. This rapidly labeled material forms an upper band when extracts of phototrophically grown cells are subjected directly to rate-zone sedimentation. In the present investigation, flash-induced absorbance changes at 605 nm have demonstrated that the upper fraction is enriched two-fold in photochemical reaction center activity when compared to chromotophores; a similar enrichment in the reaction center-associated B-875 antenna bacteriochlorophyll complex was also observed. Although b- and c-type cytochromes were present in the upper pigmented band, no photoreduction of the b-type components could be demonstrated. The endogenous c-type cytochrome (Em = +345 mV) was photooxidized slowly upon flash illumination. The extent of the reaction was increased markedly with excess exogenous ferrocytochrome c but only slightly in chromatophores. Only a small light-induced carotenoid band shift was observed. These results indicate that the rapidly labeled fraction contains photochemically competent reaction centers associated loosely with c-type and unconnected to b-type cytochrome. It is suggested that this fraction arises from new sites of cytoplasmic membrane invagination which fragment to form leaky vesicles upon cell disruption.     

A new iron-oxidizing/O2-reducing supercomplex spanning both inner and outer membranes, isolated from the extreme acidophile Acidithiobacillus ferrooxidans

The iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans involves various metalloenzymes. Here we demonstrate that the oxygen reduction pathway from ferrous iron (named downhill pathway) is organized as a supercomplex constituted of proteins located in the outer and inner membranes as well as in the periplasm. For the first time, the outer membrane-bound cytochrome c Cyc2 was purified, and we showed that it is responsible for iron oxidation and determined that its redox potential is the highest measured to date for a cytochrome c. The organization of metalloproteins inside the supramolecular structure was specified by protein-protein interaction experiments. The isolated complex spanning the two membranes had iron oxidase as well as oxygen reductase activities, indicating functional electron transfer between the first iron electron acceptor, Cyc2, and the Cu(A) center of cytochrome c oxidase aa(3). This is the first characterization of a respirasome from an acidophilic bacterium. In Acidithiobacillus ferrooxidans,O(2) reduction from ferrous iron must be coupled to the energy-consuming reduction of NAD(+)(P) from ferrous iron (uphill pathway) required for CO(2) fixation and other anabolic processes. Besides the proteins involved in the O(2) reduction, there were additional proteins in the supercomplex, involved in uphill pathway (bc complex and cytochrome Cyc(42)), suggesting a possible physical link between these two pathways.     

Cyc2p, a membrane-bound flavoprotein involved in the maturation of mitochondrial c-type cytochromes

Mitochondrial apocytochrome c and c1 are converted to their holoforms in the intermembrane space by attachment of heme to the cysteines of the CXXCH motif through the activity of assembly factors cytochrome c heme lyase and cytochrome c1 heme lyase (CCHL and CC1HL). The maintenance of apocytochrome sulfhydryls and heme substrates in a reduced state is critical for the ligation of heme. Factors that control the redox chemistry of the heme attachment reaction to apocytochrome c are known in bacteria and plastids but not in mitochondria. We have explored the function of Cyc2p, a candidate redox cytochrome c assembly component in yeast mitochondria. We show that Cyc2p is required for the activity of CCHL toward apocytochrome c and c1 and becomes essential for the heme attachment to apocytochrome c1 carrying a CAPCH instead of CAACH heme binding site. A redox function for Cyc2p in the heme lyase reaction is suggested from 1) the presence of a noncovalently bound FAD molecule in the C-terminal domain of Cyc2p, 2) the localization of Cyc2p in the inner membrane with the FAD binding domain exposed to the intermembrane space, and 3) the ability of recombinant Cyc2p to carry the NADPH-dependent reduction of ferricyanide. We postulate that, in vivo, Cyc2p interacts with CCHL and is involved in the reduction of heme prior to its ligation to apocytochrome c by CCHL.     

Photosynthetic membrane development in Rhodopseudomonas sphaeroides. Spectral and kinetic characterization of redox components of light-driven electron flow in apparent photosynthetic membrane growth initiation sites

The kinetics of light-driven electron flow and the nature of redox centers at apparent photosynthetic membrane growth initiation sites in Rhodopseudomans sphaeroides were compared to those of intracytoplasmic photosynthetic membranes. In sucrose gradients, these membrane growth sites sediment more slowly than intracytoplasmic membrane-derived chromatophores and form an upper pigmented band. Cytochromes c1, c2, b561, and b566 were demonstrated in the upper fraction by redox potentiometry; c-type cytochromes were also detected electrophoretically. Signals characteristic of light-induced reaction center bacteriochlorophyll triplet and photooxidized reaction center bacteriochlorophyll dimer states were observed by EPR spectroscopy but the Rieske iron-sulfur signal of the ubiquinol-cytochrome c2 oxidoreductase was present at a 3-fold reduced level on a reaction center basis in comparison to chromatophores. Flash-induced absorbance measurements of the upper pigmented fraction demonstrated reaction center primary and secondary semiquinone anion acceptor signals, but cytochrome b561 photoreduction and cytochrome c1/c2 reactions occurred at slow rates. This fraction was enriched approximately 2- and 4-fold in total b- and c-type cytochromes, respectively, per reaction center over chromatophores, but photoreducible b-type cytochrome was lower. Measurements of respiratory activity indicated a 1.6-fold higher level of succinate-cytochrome c oxidoreductase/reaction center than in chromatophores, but the apparent turnover rates in both preparations were low. Overall, the results suggest that complete cycles of rapid, light-driven electron flow do not occur merely by introduction of newly synthesized reaction centers into respiratory membrane, but that subsequent synthesis and assembly of appropriate components of the ubiquinol-cytochrome c2 oxidoreductase is required.     

MtrC, an outer membrane decahaem c cytochrome required for metal reduction in Shewanella putrefaciens MR-1

Shewanella putrefaciens is a facultative anaerobe that can use metal oxides as terminal electron acceptors during anaerobic respiration. Two proteins, MtrB and Cct, have been identified that are specifically involved in metal reduction. Analysis of S. putrefaciens mutants deficient in metal reduction led to the identification of two additional proteins that are involved in this process. MtrA is a periplasmic decahaem c-type cytochrome that appears to be part of the electron transport chain, which leads to Fe(III) and Mn(IV) reduction. MtrC is an outer membrane decahaem c-type cytochrome that appears to be required for the activity of the terminal Fe(III) reductase. Membrane fractions of mutants deficient in MtrC exhibited a decreased level of Fe(III) reduction compared with the wild type. We suggest that MtrC may be a component of the terminal reductase or may be required for its assembly.     

Structure,organization and expression of the genes encoding mitochondrial cytochrome c(1) and the Rieske iron-sulfur protein in Chlamydomonas reinhardtii

The sequence and organization of the Chlamydomonas reinhardtii genes encoding cytochrome c(1) ( Cyc1) and the Rieske-type iron-sulfur protein ( Isp), two key nucleus-encoded subunits of the mitochondrial cytochrome bc(1) complex, are presented. Southern hybridization analysis indicates that both Cyc1 and Isp are present as single-copy genes in C. reinhardtii. The Cyc1 gene spans 6404 bp and contains six introns, ranging from 178 to 1134 bp in size. The Isp gene spans 1238 bp and contains four smaller introns, ranging in length from 83 to 167 bp. In both genes, the intron/exon junctions follow the GT/AG rule. Internal conserved sequences were identified in only some of the introns in the Cyc1 gene. The levels of expression of Isp and Cyc1 genes are comparable in wild-type C. reinhardtii cells and in a mutant strain carrying a deletion in the mitochondrial gene for cytochrome b (dum-1). Nevertheless, no accumulation of the nucleus-encoded cytochrome c(1) or of core proteins I and II was observed in the membranes of the respiratory mutant. These data show that, in the green alga C. reinhardtii, the subunits of the cytochrome bc(1) complex fail to assemble properly in the absence of cytochrome b.     

Physiological role of glucoside 3-dehydrogenase and cytochrome c551 in the sugar oxidizing system of Flavobacterium saccharophilum

Flavobacterium saccharophilum cytoplasmic membranes contain several cytochromes linked to the respiratory chain. The presence of c-type cytochrome, cytochrome o, and a small amount of a-type cytochrome was proved. Cytochrome c551 was purified to electrophoretic homogeneity by ion-exchange chromatography and gel filtration from a membrane fraction of F. saccharophilum and its properties determined. Cytochrome c551 possessed absorption peaks at 407 nm in the oxidized form, and at 415, 521, 551 nm in the reduced form. The cytochrome c551 had a molecular weight of 15,500 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Glucoside 3-dehydrogenase of F. saccharophilum reduced the cytochrome c551 with methyl-alpha-D-glucoside, D-glucose, sucrose, or validoxylamine A. When the purified glucoside 3-dehydrogenase was incubated with methyl-alpha-D-glucoside and purified ferricytochrome c551, methyl-alpha-D-3-ketoglucoside was formed as indicated by GC-MS analysis. The addition of a substrate to the membrane fraction caused an increase in the rate of oxygen uptake and an abrupt reduction in cytochrome c551. The electron transfer in the 3-keto sugar forming system may be as follows: sugars----glucoside 3-dehydrogenase----cytochrome c551----cytochrome oxidase----O2. Thus, the electron acceptor of glucoside 3-dehydrogenase is possibly connected to the membrane-bound cytochrome system.     

NADH-dependent aryl hydrocarbon hydroxylase in rat liver mitochondrial outer membrane.

NADH-dependent 3,4-benzpyrene hydroxylase activity was detected in the purified mitochondrial outer membrane fraction from the livers of rats treated with 3-methylcholanthrene. The specific activity in the outer membrane fraction is nearly equal to that of microsomes, a level too high to be accounted for only by the microsomal contamination. On the other hand, the NADPH-dependent 3,4-benzpyrene hydroxylase activity in the outer membrane fraction is about 50% of that of microsomes. The ratio of the specific activity of NADPH- to NADH-dependent 3,4-benzpyrene hydroxylase in microsomal fraction was about 3.5, while that of the outer membrane fraction was about 1.5. Moreover, it was found that NADH-dependent 3,4-benzpyrene hydroxylase activity in mitochondrial outer membrane from control rat liver was cyanide-insensitive, while that in microsomes was cyanide-sensitive. These results suggest the presence in the mitochondrial outer membrane fraction of aryl hydrocarbon hydroxylase activity which uses as electron donor NADH nearly to the same extent as NADPH. The hydroxylase system is composed of cyanide-insensitive cytochrome P-450 and is inducible markedly by 3-methylcholanthrene treatment. The probable electron transfer pathways in the mitochondrial outer membrane cytochrome P-450 oxidase system are discussed.     

Localization of dehydrogenases, reductases, and electron transfer components in the sulfate-reducing bacterium Desulfovibrio gigas.

Various dehydrogenases, reductases, and electron transfer proteins involved in respiratory sulfate reduction by Desulfovibrio gigas have been localized with respect to the periplasmic space, membrane, and cytoplasm. This species was grown on a lactate-sulfate medium, and the distribution of enzyme activities and concentrations of electron transfer components were determined in intact cells, cell fractions prepared with a French press, and lysozyme spheroplasts. A significant fraction of formate dehydrogenase was demonstrated to be localized in the periplasmic space in addition to hydrogenase and some c-type cytochrome. Cytochrome b, menaquinone, fumarate reductase, and nitrite reductase were largely localized on the cytoplasmic membrane. Fumarate reductase was situated on the inner aspect on the membrane, and the nitrite reductase appeared to be transmembraneous. Adenylylsulfate reductase, bisulfite reductase (desulfoviridin), pyruvate dehydrogenase, and succinate dehydrogenase activities were localized in the cytoplasm. Significant amounts of hydrogenase and c-type cytochromes were also detected in the cytoplasm. Growth of D. gigas on a formate-sulfate medium containing acetate resulted in a 10-fold increase in membrane-bound formate dehydrogenase and a doubling of c-type cytochromes. Growth on fumarate with formate resulted in an additional increase in b-type cytochrome compared with lactate-sulfate-grown cells.     

Identification and characterization of UndAHRCR-6, an outer membrane endecaheme c-type cytochrome of Shewanella sp. strain HRCR-6

UndA(HRCR-6) was identified from the metal-reducing bacterium Shewanella sp. strain HRCR-6. Both in vivo and in vitro characterization results indicate that UndA(HRCR-6) is an outer membrane endecaheme c-type cytochrome and probably has a key functional role in the extracellular reduction of iron [Fe(III)] oxides and uranium [U(VI)] by Shewanella sp. HRCR-6.