Mixed-valence cytochrome oxidase-formate complex. A steady-state intermediate.
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ABSTRACT:
SUBMITTER: Brittain T
PROVIDER: S-EPMC1183699 | biostudies-other | 1977 Dec
REPOSITORIES: biostudies-other
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Project description:Elucidating the structures of intermediates in the reduction of O2 to water by cytochrome c oxidase is crucial to understanding both oxygen activation and proton pumping by the enzyme. In the work here, the reaction of O2 with the mixed-valence enzyme, in which only heme a3 and CuB in the binuclear center are reduced, has been followed by time-resolved resonance Raman spectroscopy. The results show that O==O bond cleavage occurs within the first 200 micros after reaction initiation; the presence of a uniquely stable Fe---O---O(H) peroxy species is not detected. The product of this rapid reaction is a heme a3 oxoferryl (FeIV==O) species, which requires that an electron donor in addition to heme a3 and CuB must be involved. The available evidence suggests that the additional donor is an amino acid side chain. Recent crystallographic data [Yoshikawa, S., Shinzawa-Itoh, K., Nakashima, R., Yaono, R., Yamashita, E., Inoue, N., Yao, M., Fei, M. J., Libeu, C. P., Mizushima, T., et al. Science, in press; Ostermeier, C., Harrenga, A. , Ermler, U. & Michel, H. (1997) Proc. Natl. Acad. Sci. USA 94, 10547-10553] show that one of the CuB ligands, His240, is cross-linked to Tyr244 and that this cross-linked tyrosyl is ideally positioned to participate in dioxygen activation. We propose a mechanism for O---O bond cleavage that proceeds by concerted hydrogen atom transfer from the cross-linked His---Tyr species to produce the product oxoferryl species, CuB2+---OH-, and the tyrosyl radical. This mechanism provides molecular structures for two key intermediates that drive the proton pump in oxidase; moreover, it has clear analogies to the proposed O---O bond forming chemistry that occurs during O2 evolution in photosynthesis.
| S-EPMC20922 | biostudies-other
Project description:1. The results of non-linear optimization studies on the mechanism of reaction of ferricyanide-pretreated mixed-valence-state cytochrome oxidase with O2 at 173 K are presented. The analysis is carried out on data obtained by means of dual-wavelength multi-channel spectroscopy at four wavelength pairs (444-463 nm, 604-630 nm, 608-630 nm and 830-940 nm) and at two O2 concentrations (360 micron and 520 micron). The only model that satisfies the triple requirement of a standard deviation within the standard error of the experimental data, a random distribution of residuals and good determination of the optimized parameters, is a three-intermediate sequential mechanism. 2. On the basis of the optimized values of the relative absorption coefficients of the intermediates at each wavelength obtained from the present paper together with data from optical wavelength scanning and e.p.r. spectroscopy obtained by low-temperature trapping studies, the possible valence states of the metal centres in each of the intermediates are discussed.
| S-EPMC1185846 | biostudies-other
Project description:The spin states of the haem components of mixed-valence cytochrome oxidase were studied at room temperature and at temperature down to 20K by using magnetic circular dichroism. The room-temperature studies show the presence of a low-spin ferrous haem together with a low-spin ferric haem, which we attribute to heams a3 and a respectively. At temperatures below 100K it appears that the CO of the mixed-valence CO complex may be irreversibly photolysed, and that in this case haems a and a3 assume their high-spin states. Thus in this enzyme haem-haem interactions appear possible at temperatures below 100K.
| S-EPMC1164187 | biostudies-other
Project description:Some spectra of Pseudomonas cytochrome oxidase are reported, both for comparison with those of other workers and to illustrate the differences between the ascorbate- and dithionite-reduced forms of the enzyme. A spectrum of the reduced enzyme-CO complex, prepared in the absence of added reductants by incubation under CO, is also included. Ultracentrifugation studies yielded a value for the sedimentation coefficient (s20,w) of 7.5S, and an isoelectric point of pH6.9 was determined by isoelectric focusing. Steady-state kinetic constants of the electron donors, quinol, sodium ascorbate, reduced Pseudomonas azurin and Pseudomonas ferrocytochrome c551 were investigated giving Km values of 30mM, 4mM, 49muM and 5.6muM respectively. The two protein substrates were observed to be subject to product inhibition and the Ki for oxidized Pseudomonas azurin was evaluated at 4.9muM. Steady-state kinetics were also used to investigate the effects of the oxidation products of dithionite on the oxidase and nitrite reductase activities of Pseudomonas cytochrome oxidase. These experiments showed that whereas the oxidase activity was inhibited, the nitrite reductase activity was slightly enhanced.
| S-EPMC1163870 | biostudies-other
Project description:Cytochrome P450cam is an archetypal example of the vast family of heme monooxygenases and serves as a model for an enzyme that is highly specific for both its substrate and reductase. During catalysis, it undergoes significant conformational changes of the F and G helices upon binding its substrate and redox partner, putidaredoxin (Pdx). Recent studies have shown that Pdx binding to the closed camphor-bound form of ferric P450cam results in its conversion to a fully open state. However, during catalytic turnover, it remains unclear whether this same conformational change also occurs or whether it is coupled to the formation of the critical compound I intermediate. Here, we have examined P450cam bound simultaneously by camphor, CN-, and Pdx as a mimic of the catalytically competent ferrous oxy-P450cam-Pdx state. The combined use of double electron-electron resonance and molecular dynamics showed direct observation of intermediate conformational states of the enzyme upon CN- and subsequent Pdx binding. This state is coupled to the movement of the I helix and residues at the active site, including Arg-186, Asp-251, and Thr-252. These movements enable occupation of a water molecule that has been implicated in proton delivery and peroxy bond cleavage to give compound I. These findings provide a detailed understanding of how the Pdx-induced conformational change may sequentially promote compound I formation followed by product release, while retaining stereoselective hydroxylation of the substrate of this highly specific monooxygenase.
| S-EPMC7185884 | biostudies-literature
Project description:Compound C2 is a product of the reaction of O2 and the mixed-valence state of cytochrome oxidase. The mixed-valence state of membrane-bound cytochrome oxidase is obtained at -24 degrees C, by using either ferricyanide or yeast peroxidase complex ES as oxidants, and the configurations of oxidized haem a and its associated copper (a3+Cua2+) and of reduced haem a3 and its associated copper (ac3+.CO.Cua3+) are obtained. The mixed-valence-state cytochrome oxidase mixed with O2 at -24 degrees C and flash-photolysed at -60 to -100 degrees C reacts with O2 and initially forms an oxy compound (A2) similar to that formed from the fully reduced state (A1). Thereafter the course of the reaction differs from that obtained in the fully reduced state, and absorbance increases are observed at 740--750 nm and 609 nm and a decrease at 444 nm, with no increase in absorbance at 655 nm. One possible attribution of the absorbance increases is to charge-transfer interaction between the iron of haem a3 and the copper associated with haem a3, Cua3(2+), having properties of a type-I 'blue' copper. A possible attribution of the decrease in absorbance at 444 nm is to liganding of a3(2+). A related explanation is that the 609 nm absorbance involves a charge-transfer interaction of both iron and copper as a mixed-valence binuclear complex, Cua3, having properties of a non-blue copper. Intermediates in addition to Compound C2 are not yet identifiable by chemical or spectroscopic tests. The kinetic and equilibrium properties of Compound C2 are described.
| S-EPMC1186460 | biostudies-other
Project description:The kinetics of oxidation of azurin and cytochrome c-551 catalysed by Pseudomonas aeruginosa cytochrome oxidase were re-investigated, and the steady-state parameters were evaluated by parametric and non-parametric methods. At low concentrations of substrates (e.g. less than or equal to 50 microM) the values obtained for Km and catalytic-centre activity are respectively 15 +/- 3 microM and 77 +/- 6 min-1 for azurin and 2.15 +/- 0.23 microM and 66 +/- 2 min-1 for cytochrome c-551, in general accord with previous reports assigning to cytochrome c-551 the higher affinity for the enzyme and to azurin a slightly higher catalytic rate. However, when the cytochrome c-551 concentration was extended well beyond the value of Km, the initial velocity increased, and eventually almost doubled at a substrate concentration greater than or equal to 100 microM. This result suggests a 'half-hearted' behaviour, since at relatively low cytochrome c-551 concentrations only one of the two identical binding sites of the dimeric enzyme seems to be catalytically active, possibly because of unfavourable interactions influencing the stability of the Michaelis-Menten complex at the second site. When reduced azurin and cytochrome c-551 are simultaneously exposed to Ps. aeruginosa cytochrome oxidase, the observed steady-state oxidation kinetics are complex, as expected in view of the rapid electron transfer between cytochrome c-551 and azurin in the free state. In spite of this complexity, it seems likely that a mechanism involving a simple competition between the two substrates for the same active site on the enzyme is operative. Addition of a chemically modified and redox inactive form of azurin (Hg-azurin) had no effect on the initial rate of oxidation of either azurin and cytochrome c-551, but clearly altered the time course of the overall process by removing, at least partially, the product inhibition. The results lead to the following conclusions: (i) reduced azurin and cytochrome c-551 bind at the same site on the enzyme, and thus compete; (ii) Hg-azurin binds at a regulatory site, competing with the product rather than the substrate; (iii) the two binding sites on the dimeric enzyme, though intrinsically equivalent, display unfavourable interactions. Since water is the product of the reduction of oxygen, point (iii) has important implications for the reaction mechanism.
| S-EPMC1152686 | biostudies-other
Project description:Methanogens catalyze the critical, methane-producing step (called methanogenesis) in the anaerobic decomposition of organic matter and have applications in carbon-neutral fuel production. Here, we present the first predictive model of global gene regulation of methanogenesis in a hydrogenotrophic methanogen, Methanococcus maripaludis. We generated a comprehensive list of genes (protein-coding and non-coding) for M. maripaludis through integrated analysis of the transcriptome structure and a newly constructed Peptide Atlas. The environment and gene-regulatory influence network (EGRIN) model of the strain was constructed from a compendium of transcriptome data that was collected over 100 different steady-state and time course experiments that were performed in chemostats, or batch cultures, under a spectrum of environmental perturbations that modulated methanogenesis. We discovered that at least five regulatory mechanisms act in a combinatorial scheme to inter-coordinate key steps of methanogenesis with different processes such as motility, ATP biosynthesis, and carbon assimilation. Through a combination of genetic and environmental perturbation experiments we have validated the EGRIN-predicted role of two novel TFs in the regulation of phosphate-dependent repression of formate dehydorgenase -- a key enzyme in the methanogenesis pathway.
2013-09-16 | GSE42115 | GEO
Project description:Titration of cyanide-incubated cytochrome c oxidase (ox heart cytochrome aa3) with ferrocytochrome c or with NNN'N'-tetramethyl-p-phenylenediamine initially introduces two reducing equivalents per mol of cytochrome aa3. The first equivalent reduces the cytochrome a haem iron; the second reducing equivalent is not associated with reduction of the 830 nm chromophores (e.p.r.-detectable copper) but is probably required for reduction of the e.p.r.-undetectable copper. Excess reductant introduces a third reducing equivalent into the cyanide complex of cytochrome aa3. During steady-state respiration in the presence of cytochrome c and ascorbate, the 830 nm chromophore is almost completely oxidized. It is reduced more slowly than cytochrome a on anaerobiosis. In the presence of formate or azide, some reduction at 830 nm can be seen in the steady state; in an oxygen-pulsed system, a decrease in steady-state reduction of cytochromes c and a is associated with ab increased reduction of the 830 nm species. In the formate-inhibited system the reduction of a3 on anaerobiosis shows a lag phase, the duration of which corresponds to the time taken for the 830 nm species to be reduced. It is concluded that the e.p.r.-undetectable copper (CuD) is reduced early in the reaction sequence, whereas the detectable copper (CUD) is reduced late. The latter species is probably that responsible for reduction of the cytochrome a3 haem. The magnetic association between undetectable copper and the a3 haem may not imply capability for electron transfer, which occurs more readily between cytochrome a3 and the 830 nm species.
| S-EPMC1158267 | biostudies-other
Project description:Methanogens catalyze the critical, methane-producing step (called methanogenesis) in the anaerobic decomposition of organic matter and have applications in carbon-neutral fuel production. Here, we present the first predictive model of global gene regulation of methanogenesis in a hydrogenotrophic methanogen, Methanococcus maripaludis. We generated a comprehensive list of genes (protein-coding and non-coding) for M. maripaludis through integrated analysis of the transcriptome structure and a newly constructed Peptide Atlas. The environment and gene-regulatory influence network (EGRIN) model of the strain was constructed from a compendium of transcriptome data that was collected over 100 different steady-state and time course experiments that were performed in chemostats, or batch cultures, under a spectrum of environmental perturbations that modulated methanogenesis. We discovered that at least five regulatory mechanisms act in a combinatorial scheme to inter-coordinate key steps of methanogenesis with different processes such as motility, ATP biosynthesis, and carbon assimilation. Through a combination of genetic and environmental perturbation experiments we have validated the EGRIN-predicted role of two novel TFs in the regulation of phosphate-dependent repression of formate dehydorgenase -- a key enzyme in the methanogenesis pathway. The strain was grown by continuous culture in a one-liter fermenter (New Brunswick Scientific, Edison, NJ) at 37M-BM-0C (FEMS Microbiol Lett 238: 85-91, 2004). Medium and gas compositions were modified from those for non-limiting conditions (BMC Microbiol 9: 149, 2009). The medium contained 380 mM sodium formate. The standard gassing regime was 110 mL/min H2, 40 mL/min CO2, 35 mL/min Ar, and 15 mL/min H2S/Ar mixture (1:99). Hydrogen limited condition was 21 mL/min H2. Culture samples (1.5 mL) were rapidly removed from the vessels by syringe and cell pellets collected by microcentrifugation, immediately frozen in an ethanol-dry ice bath, and stored at -80M-BM-0C. Total RNA from each sample was compared against a reference RNA pool that was generated in bulk from a mid-log phase culture of MM901. Total RNA from samples and reference were directly labeled with Cy3 or Cy5, and were hybridized to the tiling array. After hybridization and washing according to array manufacturer's instructions, the arrays were scanned by Microarray Scanner (Agilent Technologies, Santa Clara, CA). Dye-flip experiments were done for each sample.
2013-09-16 | E-GEOD-42115 | biostudies-arrayexpress