Pathways of cytochrome c oxidation by soluble and membrane-bound cytochrome aa3

In media of low ionic strength, membraneous cytochrome c oxidase, isolated cytochrome c oxidase, and proteoliposomal cytochrome c oxidase each bind cytochrome c at two sites, one of low affinity (1 microM greater than Kd' greater than 0.2 microM) and readily reversible and the other of high affinity (0.01 microM greater than Kd) and weakly reversible. When cytochrome c occupies both sites, including the low affinity site, the maximal turnover measured polarographically with ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) is independent of TMPD concentration, and lies between 250 and 400 s-1 (30 degrees C, pH 7.4) for fully activated systems. The apparent affinity of the enzyme for cytochrome c is, however, TMPD dependent. When cytochrome c occupies only the high-affinity site, the maximal turnover is closely dependent upon the concentration of TMPD, which, unlike ascorbate, can reduce bound cytochrome c. As TMPD concentration is increased, the maximal turnover approaches that seen when both sites as occupied. The lower activity of isolated cytochrome aa3 is due to the presence of inactive or inaccessible enzyme molecules. Incorporation of isolated enzyme into phospholipid vesicles restores full activity to all the subsequently accessible cytochrome aa3 molecules. Negatively charged (asolectin) vesicles show a higher cytochrome c affinity at the low-affinity sites than do the other enzyme preparations. A model for the cytochrome c-cytochrome aa3 complexes is put forward in which both sites, when occupied, are fully catalytically competent, but in which occupation of the "tight" site by a catalytically functional cytochrome c molecule is required for overall oxidation of cytochrome c via the "loose" site.     

Characterization of a new membrane-bound cytochrome c of Rhodopseudomonas capsulata

A cytochrome c (cyt. c) was solubilized with Triton-X-100 and co-purified with cytochrome c oxidase from membranes of chemotrophically grown cells of Rhodopseudomonas capsulata. Cyt. c and cytochrome oxidase were separated on Sephadex G-50 columns. Antibodies against cytochrome c2 from the same bacterium did not cross react with the membrane-bound cyt. c. The IEP of the membrane-bound cyt. c was found to be pH 8.2, the midpoint potential was 234 +/- 11 mV at pH 7.0. This cyt. c binds CO. The native cyt. c is a dimer with an apparent Mr of 25000 containing 2 mol heme per mol dimer, which is believed to function as an electron donor for the high-potential cytochrome c oxidase.     

A bacterial cytochrome c heme lyase. CcmF forms a complex with the heme chaperone CcmE and CcmH but not with apocytochrome c.

Biogenesis of c-type cytochromes in Escherichia coli involves a number of membrane proteins (CcmA-H), which are required for the transfer of heme to the periplasmically located apocytochrome c. The pathway includes (i) covalent, transient binding of heme to the periplasmic domain of the heme chaperone CcmE; (ii) the subsequent release of heme; and (iii) transfer and covalent attachment of heme to apocytochrome c. Here, we report that CcmF is a key player in the late steps of cytochrome c maturation. We demonstrate that the conserved histidines His-173, His-261, His-303, and His-491 and the tryptophan-rich signature motif of the CcmF protein family are functionally required. Co-immunoprecipitation experiments revealed that CcmF interacts directly with the heme donor CcmE and with CcmH but not with apocytochrome c. We propose that CcmFH forms a bacterial heme lyase complex for the transfer of heme from CcmE to apocytochrome c.     

Structure of the copper sites in membrane-bound cytochrome c oxidase

The structures of membrane proteins are difficult to obtain by crystallography and may be altered by the detergents used in their extraction. X-ray absorption spectroscopy has been used to identify the structures of the copper atoms of the membrane-bound enzyme in mitochondria and in submitochondrial particles at respective concentrations of 100 and 200 micron of molar copper. To within the experimental error, the x-ray absorption spectra of the copper atoms of the membrane-bound and the Yonetani (Yonetani, T. (1961) J. Biol. Chem. 236, 1680-1688) purified oxidase are identical; all detectable shells of the active site indicate no alteration of structural parameters. Significant differences are found when compared to the Hartzell-Beinert (Hartzell, R. C., and Beinert, H. (1974) Biochim. Biophys. Acta 368, 318-338) preparation. Extended x-ray absorption fine structure technology is now adequate for the direct studies of membrane proteins in situ in their natural environment.     

The cellular location and specificity of bacterial cytochrome c peroxidases.

We have found that an anti-CD11c monoclonal antibody (MAb) inhibits the respiratory burst induced in phorbol 12-myristate 13-acetate (PMA)-differentiated U937 cells as well as in human peripheral blood monocytes and neutrophils upon cell stimulation with concanavalin A. The MAb had no effect, however, when the added stimulus was fMet-Leu-Phe or PMA. Flow cytometry analyses indicated that concanavalin A was able to interact with CD11c. The anti-CD11c MAb inhibited significantly concanavalin A binding to differentiated U937 cells, and concanavalin A blocked binding of anti-CD11c MAb to the cells. Binding of labelled concanavalin A to membrane proteins which were separated by PAGE and transferred to nitrocellulose paper indicated that proteins with apparent molecular masses similar to those of CD11c (150 kDa) and CD18 (95 kDa) molecules were the main concanavalin A-binding proteins in differentiated U937 cells as well as in mature neutrophils. Similar experiments carried out in the presence of the anti-CD11c MAb showed a specific and significant inhibition of concanavalin A binding to the CD11c molecule. These results indicate that concanavalin A binds to the CD11c molecule and this binding is responsible for the concanavalin A-induced respiratory burst in PMA-differentiated U937 cells as well as in human mature monocytes and neutrophils.     

The role of cytochrome c4 in bacterial respiration. Cellular location and selective removal from membranes.

The cellular location of cytochrome c4 in Pseudomonas stutzeri and Azotobacter vinelandii was investigated by the production of spheroplasts. Soluble cytochrome c4 was found to be located in the periplasm in both organisms. The remaining cytochrome c4 was membrane-bound. The orientation of this membrane-bound cytochrome c4 fraction was investigated by proteolysis of the cytochrome on intact spheroplasts. In P. stutzeri, 78% of the membrane-bound cytochrome c4 could be proteolysed, whilst 82% of the spheroplasts remained intact, suggesting that the membrane-bound cytochrome c4 is on the periplasmic face of the membrane in this organism. Cytochrome c4 was not susceptible to proteolysis on A. vinelandii spheroplasts, in spite of being digestible in the purified state. Cytochrome c5 was shown to have a similar cellular distribution to cytochrome c4. Selective removal of cytochrome c4 from membranes of P. stutzeri was accomplished by the use of sodium iodide and propan-2-ol, with the retention of most of the ascorbate-TMPD (NNN'N'-tetramethylbenzene-1,4-diamine) oxidase activity associated with the membrane. Sodium iodide removed most of the cytochrome c4 from A. vinelandii membranes with retention of 62% of the ascorbate-TMPD oxidase activity. Cytochrome c4 could be returned to the washed membranes, but with no recovery of this enzyme activity. We conclude that cytochrome c4 is not involved in the ascorbate-TMPD oxidase activity associated with the membranes of these two organisms.     

Arrangement of the subunits in solubilized and membrane-bound cytochrome c oxidase from bovine heart.

The arrangement of the six cytochrome c oxidase subunits in the inner membrane of bovine heart mitochondria was investigated. The experiments were carried out in three steps. In the first step, exposed subunits were coupled to the membrane-impermeant reagent p-diazonium benzene [32S]sulfonate. In the second step, the membranes were lysed with cholate anc cytochrome c oxidase was isolated by immunoprecipitation. In the third step, the six cytochrome c oxidase subunits were separated from each other by dodecyl sulfate-acrylamide gel electrophoresis and scanned for radioactivity. Exposed subunits on the outer side of the mitochondrial inner membrane were identified by labeling intact mitochondria. Exposed subunits on the matrix side of the inner membrane were identified by labeling sonically prepared submitochondrial particles in which the matrix side of the inner membrane is exposed to the suspending medium. Since sonic irradiation leads to a rearrangement of cytochrome c oxidase in a large fraction of the resulting submitochondrial particles, an immunochemical procedure was developed for isolating particles with a low content of displaced cytochrome c oxidase. With mitochondria, subunits II, V, and VI were labeled, whereas in purified submitochondrial particles most of the label was in subunit III. The arrangement of cytochrome c oxidase in the mitochondrial inner membrane is thus transmembraneous and asymmetric; subunits II, V, and VI are situated on the outer side, subunit III is situated on the matrix side, and subunits I and IV are buried in the interior of the membrane. In a study of purified cytochrome c oxidase labeled with p-diazonium benzene [32S]sulfonate, the results were similar to those obtained with the membrane-bound enzyme. Subunits I and IV were inaccessible to the reagent, whereas the other four subunits were accessible. In contrast, all six subunits became labeled if the enzyme was dissociated with dodecyl sulfate before being exposed to the labeling reagent.     

Calcium-dependent heme structure in the reduced forms of the bacterial cytochrome c peroxidase from Paracoccus pantotrophus

This work reports for the first time a resonance Raman study of the mixed-valence and fully reduced forms of Paracoccus pantotrophus bacterial cytochrome c peroxidase. The spectra of the active mixed-valence enzyme show changes in the structure of the ferric peroxidatic heme compared to the fully oxidized enzyme; these differences are observed upon reduction of the electron-transferring heme and upon full occupancy of the calcium site. For the mixed-valence form in the absence of Ca(2+), the peroxidatic heme is six-coordinate and low-spin on the basis of the frequencies of the structure-sensitive Raman lines: the enzyme is inactive. With added Ca(2+), the peroxidatic heme is five-coordinate high-spin and active. The calcium-dependent spectral differences indicate little change in the conformation of the ferrous electron-transferring heme, but substantial changes in the conformation of the ferric peroxidatic heme. Structural changes associated with Ca(2+) binding are indicated by spectral differences in the structure-sensitive marker lines, the out-of-plane low-frequency macrocyclic modes, and the vibrations associated with the heme substituents of that heme. The Ca(2+)-dependent appearance of a strong gamma 15 saddling-symmetry mode for the mixed-valence form is consistent with a strong saddling deformation in the active peroxidatic heme, a feature seen in the Raman spectra of other peroxidases. For the fully reduced form in the presence of Ca(2+), the resonance Raman spectra show that the peroxidatic heme remains high-spin.     

Free radical fragmentation of cardiolipin by cytochrome c

The effect of cytochrome c (cyt c) on degradation of cardiolipin in its polar part was investigated in cardiolipin/phosphatidylcholine (CL/PC) liposomes incubated with cyt c/H₂O₂/and (or) ascorbate by high-performance thin layer chromatography and MALDI-TOF mass spectrometry. It has been shown that phosphatidic acid (PA) and phosphatidylhydroxyacetone (PHA) were formed in the system under conditions where hydrogen peroxide favours a release of heme iron from cyt c. The formation of PA and PHA occurs via an OH-induced fragmentation taking place in the polar moiety of cardiolipin. Formation of fragmentation products correlated with the loss of CL in CL/PC liposomes incubated with cyt c/H₂O₂/ascorbate or with Cu²⁺/H₂O₂/ascorbate.     

Hydrogen exchange in a bacterial cytochrome c: a fingerprint of the cytochrome c fold

The hydrogen exchange rates of backbone amides in a minimal (71 amino acid long) monoheme cytochrome c were determined as a function of pH in the absence and in the presence of guanidinium chloride. These data permitted the identification of units undergoing the opening reaction that precedes hydrogen exchange through a common mechanism. The opening units broadly correlate with the secondary structure elements of the protein. It is found that, despite the significant difference in primary sequence, the distribution of the opening units within the three-dimensional structure of the cytochrome studied here closely resembles that determined in mitochondrial c-type cytochromes. It is proposed that the observed distribution represents a fingerprint of the cytochrome c fold and has a role in directing the folding/unfolding of the protein.     

Proton-coupled electron equilibrium in soluble and membrane-bound cytochrome c oxidase from Paracoccus denitrificans

The pH dependence of electron and proton re-equilibration upon CO photolysis from two-electron-reduced aa3 oxidase was followed by time-resolved electrometry and optical spectroscopy. Optical spectroscopy on soluble Paracoccus denitrificans enzyme at alkaline pH revealed a slow (1 ms) component of electron re-equilibration coupled to the release of protons from the catalytic site. In the work [Br?ndén, M., et al. (2003) Biochemistry 42, 13178-13184], it was proposed that this proton is released from a water molecule in the catalytic site, located deep in the membrane dielectric. Movement of charged particles such as protons across the dielectric should create an electric potential. However, recording of the time course of the potential generation did not show any potential development in the millisecond time domain, but instead, potential generation was found with an apparent time constant of 50-100 micros. This potential was generated upon proton release from the level of the binuclear catalytic site through the K-channel, because mutation in this channel abolishes the potential generation altogether. The apparent inconsistency between results from optical spectroscopy and electrometry was solved by optical experiments on the membrane-incorporated enzyme. Reconstituting the enzyme into proteoliposomes speeds up the slow electron redistribution process by a factor of 10 and shows the same time constant as potential generation. The possible mechanism of such dramatic change in the rate of proton transfer is discussed.     

A complex of cardiac cytochrome c1 and cytochrome c.

The interactions of cytochrome c1 and cytochrome c from bovine cardiac mitochondria were investigated. Cytochrome c1 and cytochrome c formed a 1:1 molecular complex in aqueous solutions of low ionic strength. The complex was stable to Sephadex G-75 chromatography. The formation and stability of the complex were independent of the oxidation state of the cytochrome components as far as those reactions studied were concerned. The complex was dissociated in solutions of ionic strength higher than 0.07 or pH exceeding 10 and only partially dissociated in 8 M urea. No complexation occurred when cytochrome c was acetylated on 64% of its lysine residues or photooxidized on its 2 methionine residues. Complexes with molecular ratios of less than 1:1 (i.e. more cytochrome c) were obtained when polymerized cytochrome c, or cytochrome c with all lysine residues guanidinated, or a "1-65 heme peptide" from cyanogen bromide cleavage of cytochrome c was used. These results were interpreted to imply that the complex was predominantly maintained by ionic interactions probably involving some of the lysine residues of cytochrome c but with major stabilization dependent on the native conformations of both cytochromes. The reduced complex was autooxidizable with biphasic kinetics with first order rate constants of 6 X 10(-5) and 5 X U0(-5) s-1 but did not react with carbon monoxide. The complex reacted with cyanide and was reduced by ascorbate at about 32% and 40% respectively, of the rates of reaction with cytochrome c alone. The complex was less photoreducible than cytochrome c1 alone. The complex exhibited remarkably different circular dichroic behavior from that of the summation of cytochrome c1 plus cytochrome c. We concluded that when cytochromes c1 and c interacted they underwent dramatic conformational changes resulting in weakening of their heme crevices. All results available would indicate that in the complex cytochrome c1 was bound at the entrance to the heme crevice of cytochrome c on the methionine-80 side of the heme crevice.     

Studies of the kinetics of oxidation of cytochrome c by cytochrome c oxidase: comparison of the reactions of purified and membrane-bound oxidase

l-Asparaginase (EC 3.5.1.1.) activity has been detected in crude extracts of Lupinus arboreus young leaves, root tips, flower buds, and developing seeds. The enzyme was also present in Lupinus angustifolius root tips, developing nodules, and developing seeds. The asparaginase from each of these tissues had the same electrophoretic mobility on polyacrylamide gels and a K_m of 6–8 mm for asparagine. In extracts other than those of the developing seeds, asparaginase activity was dependent upon the inclusion of K⁺ ion and a sulfhydryl protectant in the extraction buffer. No asparaginase activity was detected in mature leaves, in the plant fraction of nodules that were fixing nitrogen, nor in root tissue further than 1.5 cm from the root tip. Asparaginase has been purified 326- and 230-fold from L. arboreus and L. angustifolius developing seeds, respectively. A molecular weight of 75,000 was obtained by gel filtration. An apparent K_m of 6.6 and 7.0 mm for asparagine was determined for the purified L. arboreus and L. angustifolius asparaginases, respectively. Of the amides, nitriles, and hydroxamates examined, the L. arboreus enzyme hydrolyzed only l-asparagine and dl-aspartyl hydroxamate. This same enzyme was inhibited by d-asparagine, 5-diazo-4-oxo-l-norvaline, dl-aspartyl hydroxamate, d-and l-aspartate, 3-cyano-l-alanine, glycine, and cysteine. Glutamine, glutamine analogs, and a number of other amino acids, amides and amines did not inhibit the L. arboreus asparaginase.     

Free and membrane-bound chloroplast polyribosomes Chlamydomonas reinhardtii.

Over half of the chloroplast ribosomes isolated from growing cultures of Chlamydomonas reinhardtii are bound to chloroplast thylakoid membranes if completion of nascent polypeptide chains is prevented by chloramphenicol. The free chloroplast ribosomes are recovered in homogenate supernatants, and presumably originate from the chloroplast stroma. Only about 10% of these free chloroplast ribosomes are polyribosomes, even under conditions when 70% of free cytoplasm ribosomes are recovered as polyribosomes. The nonionic detergent Nonidet P-40 liberates atypical polyribosomes (Type I), from membranes, which require both ribonuclease and proteases for complete conversion to monomeric ribosomes. Thus Type I particles are held together by mRNA but are also held together by peptide bonds. These Type I polyribosomes probably are not bound to intact membrane, but might be bound to some protein-containing sub-membrane particle. The Type I polyribosomes are dissociated to ribosomal subunits by puromycin and high salt, and contained 0.2 to 1 nascent chain per ribosome. If membranes are treated with Nonidet and proteases at the same time, polyribosomes which are digested to monomeric ribosomes by ribonuclease alone (Type II) are obtained. Type II polyribosomes are smaller than Type I, and probably represent the true size distribution of polyribosomes on the membranes. At least 50% of the membrane-bound ribosomes are polyribosomes, since that much membrane bound chloroplast RNA is recovered as Type I or Type II polyribosomes.     

Multiple low spin forms of the cytochrome c ferrihemochrome. EPR spectra of various eukaryotic and prokaryotic cytochromes c.

1. Despite the same methionine-sulfur:heme-iron:imidazole-nitrogen hemochrome structure observed by x-ray crystallography in four of the seven c-type eukaryotic and prokaryotic cytochromes examined, and the occurrence of the characteristic 695 nm absorption band correlated with the presence of a methionine-sulfur:heme-iron axial ligand in all seven proteins, they fall into two distinct classes on the basis of their EPR and optical spectra. The horse, tuna, and bakers' yeast iso-1 cytochromes c have a predominant neutral pH EPR form with g1=3.06, g2=2.26, and g3=1.25, while the bakers' yeast iso-2 and Euglena cytochromes c, the Rhodospirillum rubrum cytochrome c2, and the Paracoccus denitrificans cytochrome c550 all have a predominant neutral pH EPR form with g1=3.2, g2=2.05, and g3=1.39. The ferricytochromes with g1=3.06 have a B-Q splitting that is approximately 150 cm-1 larger than the ferricytochromes with g1=3.2. 2. Each of the cytochromes displays up to four low spin EPR forms that are in pH-dependent equilibrium and can all be observed at near neutral pH. As the pH is raised the predominant neutral pH form is converted into two forms with g1=3.4 and g1=3.6, identified by comparsion with model compounds and other heme proteins as epsilon-amino:heme-iron:imidazole and bis-epsilon-amino:heme-iron ferrihemochromes, respectively. 3. The pK for the conversion of the predominant neutral pH EPR form into the alkaline pH forms is the same as the pK for the disappearance of the 695 nm absorption band for the cytochromes, even though these pK values range over 2 pH units. This confirms that the g1=3.06 and g1=3.2 forms contain the methionine-sulfur:heme-iron axial ligand while the g1=3.4 and the g1=3.6 forms do not. 4. At extremes of pH, the horse and bakers' yeast iso-1 proteins display several high and low spin forms that are identified, showing that a variety of protein-derived ligands will coordinate to the heme iron including methionine and cysteine sulfur, histidine imidazole, and lysine epsilon-amine. 5. The spectrum of horse cytochrome c with added azide, cyanide, hydroxide, or imidazole as axial ligands has also been examined. 6. From a comparison of the EPR and optical spectral characteristics of these groups of cytochromes with model compounds, it is suggested that the difference between them is due to a change in the hydrogen bonding or perhaps even in the protonation of N-1 of the heme iron-bound histidine imidazole.     

Disease-related mutations in cytochrome c oxidase studied in yeast and bacterial models.

Mitochondrial cytochrome c oxidase is a key protonmotive component of the respiratory chain. Mutations in the mitochondrially-encoded subunits of the complex have been reported in association with a range of diseases. In this work we used yeast and bacterial mutants to assess the effect of human mutations in subunit 1 (L196I) and subunit 3 (G78S, A200T, Delta F94-F98, F251L and W249Stop). While the stop mutation at the C-terminus of subunit 3 and the short deletion were highly deleterious and abolished the assembly of the mitochondrial enzyme, the four missense mutations caused little or no effect on the respiratory function. Detailed analysis of G78S, A200T and Delta F94-F98 in Rhodobacter sphaeroides confirmed and extended these observations. We show in this study that the combination of yeast and bacterial models is a useful tool to elucidate the effect of mutations in the catalytic core of cytochrome oxidase. The yeast enzyme is highly similar to the human enzyme and provides a good model to assess the deleterious effect of reported mutations. The bacterial system allows detailed biochemical analysis of the effect of the mutations on the function and assembly of the catalytic core of the enzyme.