The reduction of Pseudomonas cytochrome c551 oxidase by chromous ions.
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ABSTRACT:
SUBMITTER: Barber D
PROVIDER: S-EPMC1164743 | biostudies-other | 1977 Jun
REPOSITORIES: biostudies-other
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Project description:Cytochrome c oxidase from bovine heart binds Ca(2+) reversibly at a specific Cation Binding Site located near the outer face of the mitochondrial membrane. Ca(2+) shifts the absorption spectrum of heme a, which allowed previously to determine the kinetics and equilibrium characteristics of the binding. However, no effect of Ca(2+) on the functional characteristics of cytochrome oxidase was revealed earlier. Here we report that Ca(2+) inhibits cytochrome oxidase activity of isolated bovine heart enzyme by 50-60% with Ki of ?1 µM, close to Kd of calcium binding with the oxidase determined spectrophotometrically. The inhibition is observed only at low, but physiologically relevant, turnover rates of the enzyme (?10 s(-1) or less). No inhibitory effect of Ca(2+) is observed under conventional conditions of cytochrome c oxidase activity assays (turnover number >100 s(-1) at pH 8), which may explain why the effect was not noticed earlier. The inhibition is specific for Ca(2+) and is reversed by EGTA. Na(+) ions that compete with Ca(2+) for binding with the Cation Binding Site, do not affect significantly activity of the enzyme but counteract the inhibitory effect of Ca(2+). The Ca(2+)-induced inhibition of cytochrome c oxidase is observed also with the uncoupled mitochondria from several rat tissues. At the same time, calcium ions do not inhibit activity of the homologous bacterial cytochrome oxidases. Possible mechanisms of the inhibition are discussed as well as potential physiological role of Ca(2+) binding with cytochrome oxidase. Ca(2+)- binding at the Cation Binding Site is proposed to inhibit proton-transfer through the exit part of the proton conducting pathway H in the mammalian oxidases.
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Project description:Comparative analysis of the transcriptional response, as quantified by RNA-Seq, of Mycobacterium tuberculosis (Mtb) during inhibition of the terminal cytochrome oxidases, Cytochrome bd oxidase and Cytochrome bcc:aa3. This study was designed to evaluate the efficacy if a novel small molecule inhibitor of the Cytochrome bd oxidase. Specifically, we compared the transcriptional response of Mtb during inhibition by Q203 with a Cytochrome bd deletion mutant to the transcriptional response of Mtb treated with a combination of Q203 and the purported Cytorchomre bd small molecule inhibitor (ND-011992).
2020-10-07 | GSE159080 | GEO
Project description:Cytochrome c oxidase (CcO) catalyzes the four-electron reduction of oxygen to water, the one-electron reductant Cytochrome c (Cytc) being the source of electrons. Recently we reported a functional model of CcO that electrochemically catalyzes the four-electron reduction of O(2) to H(2)O (Collman et al. Science 2007, 315, 1565). The current paper shows that the same functional CcO model catalyzes the four-electron reduction of O(2) using the actual biological reductant Cytc in a homogeneous solution. Both single and steady-state turnover kinetics studies indicate that O(2) binding is rate-determining and that O-O bond cleavage and electron transfer from reduced Cytc to the oxidized model complex are relatively fast.
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Project description:The magnetic properties of the haem groups of Pseudomonas cytochrome oxidase and its cyanide-bound derivatives were studied in both the oxidized and reduced states by means of m.c.d. (magnetic circular dichroism) at low temperatures. In addition, the oxidized forms of the enzyme were also investigated by e.p.r. (electron-paramagnetic-resonance) spectroscopy, and a parallel study, using both e.p.r. and m.c.d., was made on Pseudomonas cytochrome c-551 to aid spectral assignments. For ascorbate-reduced Pseudomonas cytochrome oxidase, the temperature-independence of those features in the m.c.d. spectrum corresponding to the haem c, and the temperature-dependence of those signals corresponding to the haem d1, showed the former to be low-spin and the latter to be high-spin (s = 2). However, addition of cyanide to the reduced enzyme gave a form of the protein that was completely low-spin. The e.p.r. and m.c.d. sectra of oxidized Pseudomonas cytochrome oxidase and its cyanide derivative were consistent with the haem c and d1 components being low-spin in both cases. Pseudomonas cytochrome c-551 was found to be low-spin in both its oxidized and reduced redox states.
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Project description:The fully oxidized form of cytochrome c oxidase, immediately after complete oxidation of the fully reduced form, pumps protons upon each of the initial 2 single-electron reduction steps, whereas protons are not pumped during single-electron reduction of the fully oxidized "as-isolated" form (the fully oxidized form without any reduction/oxidation treatment) [Bloch D, et al. (2004) The catalytic cycle of cytochrome c oxidase is not the sum of its two halves. Proc Natl Acad Sci USA 101:529-533]. For identification of structural differences causing the remarkable functional difference between these 2 distinct fully oxidized forms, the X-ray structure of the fully oxidized as-isolated bovine heart cytochrome c oxidase was determined at 1.95-A resolution by limiting the X-ray dose for each shot and by using many (approximately 400) single crystals. This minimizes the effects of hydrated electrons induced by the X-ray irradiation. The X-ray structure showed a peroxide group bridging the 2 metal sites in the O(2) reduction site (Fe(3+)-O(-)-O(-)-Cu(2+)), in contrast to a ferric hydroxide (Fe(3+)-OH(-)) in the fully oxidized form immediately after complete oxidation from the fully reduced form, as has been revealed by resonance Raman analyses. The peroxide-bridged structure is consistent with the reductive titration results showing that 6 electron equivalents are required for complete reduction of the fully oxidized as-isolated form. The structural difference between the 2 fully oxidized forms suggests that the bound peroxide in the O(2) reduction site suppresses the proton pumping function.
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Project description:The reduction of ferricytochrome c and ferricytochrome c carboxymethylated at the haem-linked methionine (residue 80) by Cr(2+) ions was studied by stopped-flow techniques. At pH6.2 the kinetics of reduction of ferricytochrome c are simple and correspond to a second-order rate constant of 1.21x10(3)m(-1).s(-1). Under identical conditions the kinetics of reduction of the carboxymethyl derivative, carboxymethyl-cytochrome c, are complex; two Cr(2+)-concentration-dependent processes (1.5x10(4)m(-1).s(-1) and 1.3x10(3)m(-1).s(-1)) lead to the formation of an intermediate which decays in monomolecular fashion (0.15s(-1)) to form the normal fully reduced material. The kinetic difference spectrum for the overall process corresponds to that found statically, whereas the kinetic difference spectrum of the intermediate minus the oxidized form resembles that of the low-spin ferrous form of carboxymethyl-cytochrome c minus oxidized carboxymethyl-cytochrome c. A model is proposed in which the reduction of low-spin ferric carboxymethyl-cytochrome c to high-spin ferrous carboxymethyl-cytochrome c involves a low-spin ferrous intermediate. The monomolecular step involving the decay of this low-spin ferrous intermediate is associated with an activation energy of approx. 126kJ.mol(-1) and is thought to involve both a change of spin state and a protein-conformational event. Although carboxymethyl-cytochrome c represents a mixture of species separable on a charge basis, the above observations were independent of which species was chosen for study.
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Project description:Combining peroxidase activity-based heme staining (TMBZ/SDS/PAGE) with mass spectrometry analyses (Nano LC-MS/MS) of protein extracts from wild-type and appropriate mutants, we provide evidence that the polychlorinated biphenyl degrader Pseudomonas pseudoalcaligenes KF707 primarily expresses a caa3 -type cytochrome c oxidase (caa3 -Cox) using cytochrome (cyt) c4 as an electron donor in cells grown with biphenyl versus glucose as the sole carbon source. Homology modeling of KF707 caa3 -Cox using the three-dimensional structure of that from Thermus thermophilus highlights multiple similarities and differences between the proton channels in subunit I of the aa3 - and caa3 -Cox of Paracoccus and Thermus spp., respectively. To our knowledge, this is the first report demonstrating the presence of a caa3 -Cox using cyt c4 as an electron donor in a Pseudomonas species.
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Project description:Creating an artificial functional mimic of the mitochondrial enzyme cytochrome c oxidase (CcO) has been a long-term goal of the scientific community as such a mimic will not only add to our fundamental understanding of how CcO works but may also pave the way for efficient electrocatalysts for oxygen reduction in hydrogen/oxygen fuel cells. Here we develop an electrocatalyst for reducing oxygen to water under ambient conditions. We use site-directed mutants of myoglobin, where both the distal Cu and the redox-active tyrosine residue present in CcO are modelled. In situ Raman spectroscopy shows that this catalyst features very fast electron transfer rates, facile oxygen binding and O-O bond lysis. An electron transfer shunt from the electrode circumvents the slow dissociation of a ferric hydroxide species, which slows down native CcO (bovine 500 s(-1)), allowing electrocatalytic oxygen reduction rates of 5,000 s(-1) for these biosynthetic models.
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Project description:Pseudomonas pseudoalcaligenes CECT5344 grows in minimal medium containing cyanide as the sole nitrogen source. Under these conditions, an O2-dependent respiration highly resistant to cyanide was detected in cell extracts. The structural genes for the cyanide-resistant terminal oxidase, cioA and cioB, are clustered and encode the integral membrane proteins that correspond to subunits I and II of classical cytochrome bd, although the presence of heme d in the membrane could not be detected by difference spectra. The cio operon from P. pseudoalcaligenes presents a singular organization, starting upstream of cioAB by the coding sequence of a putative ferredoxin-dependent sulfite or nitrite reductase and spanning downstream two additional open reading frames that encode uncharacterized gene products. PCR amplifications of RNA (reverse transcription-PCR) indicated the cyanide-dependent up-regulation and cotranscription along the operon. The targeted disruption of cioA eliminates both the expression of the cyanide-stimulated respiratory activity and the growth with cyanide as the nitrogen source, which suggests a critical role of this cytochrome bd-related oxidase in the metabolism of cyanide by P. pseudoalcaligenes CECT5344.
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Project description:Cytochrome c oxidase is a transmembrane proton pump that builds an electrochemical gradient using chemical energy from the reduction of O(2). Ionization states of all residues were calculated with Multi-Conformation Continuum Electrostatics (MCCE) in seven anaerobic oxidase redox states ranging from fully oxidized to fully reduced. One long-standing problem is how proton uptake is coupled to the reduction of the active site binuclear center (BNC). The BNC has two cofactors: heme a(3) and Cu(B). If the protein needs to maintain electroneutrality, then 2 protons will be bound when the BNC is reduced by 2 electrons in the reductive half of the reaction cycle. The effective pK(a)s of ionizable residues around the BNC are evaluated in Rhodobacter sphaeroides cytochrome c oxidase. At pH 7, only a hydroxide coordinated to Cu(B) shifts its pK(a) from below 7 to above 7 and so picks up a proton when heme a(3) and Cu(B) are reduced. Glu I-286, Tyr I-288, His I-334, and a second hydroxide on heme a(3) all have pK(a)s above 7 in all redox states, although they have only 1.6-3.5 DeltapK units energy cost for deprotonation. Thus, at equilibrium, they are protonated and cannot serve as proton acceptors. The propionic acids near the BNC are deprotonated with pK(a)s well below 7. They are well stabilized in their anionic state and do not bind a proton upon BNC reduction. This suggests that electroneutrality in the BNC is not maintained during the anaerobic reduction. Proton uptake on reduction of Cu(A), heme a, heme a(3), and Cu(B) shows approximately 2.5 protons bound per 4 electrons, in agreement with prior experiments. One proton is bound by a hydroxyl group in the BNC and the rest to groups far from the BNC. The electrochemical midpoint potential (E(m)) of heme a is calculated in the fully oxidized protein and with 1 or 2 electrons in the BNC. The E(m) of heme a shifts down when the BNC is reduced, which agrees with prior experiments. If the BNC reduction is electroneutral, then the heme a E(m) is independent of the BNC redox state.
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