Polar Residues in Helix VIII of Subunit I of Cytochrome c Oxidase Influence the Activity and the Structure of the Active Site (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1998
Rhodobacter sphaeroides expresses a bb 3-type quinol oxidase, and two cytochrome c oxidases: cytochrome aa 3 and cytochrome cbb 3. We report here the characterization of the genes encoding this latter oxidase. The ccoNOQP gene cluster of R. sphaeroides contains four open reading frames with high similarity to all ccoNOQP/fixNOQP gene clusters reported so far. CcoN has the six highly conserved histidines proposed to be involved in binding the low spin heme, and the binuclear center metals. ccoO and ccoP code for membrane bound mono-and diheme cytochromes c. ccoQ codes for a small hydrophobic protein of unknown function. Upstream from the cluster there is a conserved Fnr/FixK-like box which may regulate its expression. Analysis of a R. sphaeroides mutant in which the ccoNOQP gene cluster was inactivated confirms that this cluster encodes the cbb 3-type oxidase previously purified. Analysis of proton translocation in several strains shows that cytochrome cbb 3 is a proton pump. We also conclude that cytochromes cbb 3 and aa 3 are the only cytochrome c oxidases in the respiratory chain of R. sphaeroides.
Journal of Bioenergetics and Biomembranes, 1993
Cytochromeaa 3 ofRhodobacter sphaeroides and cytochromebo ofE. coli are useful models of the more complex cytochromec oxidase of eukaryotes, as demonstrated by the genetic, spectroscopic, and functional studies reviewed here. A summary of site-directed mutants of conserved residues in these two enzymes is presented and discussed in terms of a current model of the structure of the metal centers and evidence for regions of the protein likely to be involved in proton transfer. The model of ligation of the hemea 3 (oro)-CuB center, in which both hemes are bound to helix X of subunit I, has important implications for the pathways and control of electron transfer.
Biochemistry, 2009
Properties of Arg481 mutants of the aa 3 -type cytochrome c oxidase from Rhodobacter sphaeroides suggest that neither R481 nor the nearby D-propionate of heme a 3 is likely to be the proton loading site of the proton pump Abstract Cytochrome c oxidase utilizes the energy from electron transfer and reduction of oxygen to water and pumps protons across the membrane, generating a proton motive force. A large body of biochemical work has shown that all the pumped protons enter the enzyme through the D-channel, which is apparent in X-ray structures as a chain of water molecules connecting D132 at the cytoplasmic surface of the enzyme, to E286, near the enzyme active site. The exit pathway utilized by pumped protons beyond this point and leading to the bacterial periplasm is not known. Also not known is the proton loading site (or sites) which undergoes changes in pK a in response to the chemistry at the enzyme active site and drives the proton pump mechanism. In this paper we examine the role of R481, a highly conserved arginine that forms an ion pair with the D-propionate of heme a 3 . The R481H, R481N, R481Q and R481L mutants were examined. The R481H mutant oxidase is about 18% active and pumps protons with about 40% of the stoichiometry of the wild type. The R481N, R481Q and R481L mutants each retain only about 5% of the steady state activity, and this is shown to be due to inhibition of steps in the reaction of O 2 with the reduced enzyme. Neither the R481N nor the R481Q mutant oxidases pump protons but, remarkably, the R481L mutant does pump protons with the same efficiency as the R481H mutant. Since the proton pump is clearly operating in the R481L mutant, these results rule out an essential role in the proton pump mechanism for R481 or its hydrogen bond partner, the D-propionate of heme a 3 .
Proceedings of the National Academy of Sciences, 2006
The heme-copper oxidases constitute a superfamily of terminal dioxygen-reducing enzymes located in the inner mitochondrial or in the bacterial cell membrane. The presence of a mechanistically important covalent bond between a histidine ligand of the copper ion (Cu B) in the active site and a generally conserved tyrosine residue nearby has been shown to exist in the canonical cytochrome c oxidases. However, according to sequence alignment studies, this critical tyrosine is missing from the subfamily of cbb3-type oxidases found in certain bacteria. Recently, homology modeling has suggested that a tyrosine residue located in a different helix might fulfill this role in these enzymes. Here, we show directly by methods of protein chemistry and mass spectrometry that there is indeed a covalent link between this tyrosine and the copper-ligating histidine. The identity of the cross-linked tyrosine was determined by showing that the cross-link is not formed when this residue is replaced by phenylalanine, even though structural integrity is maintained. These results suggest a universal functional importance of the histidine-tyrosine cross-link in the mechanism of O2 reduction by all heme-copper oxidases. mass spectrometry
Spectroscopic and genetic evidence for two heme-Cu-containing oxidases in Rhodobacter sphaeroides
Journal of Bacteriology, 1992
It has recently become evident that many bacterial respiratory oxidases are members of a superfamily that is related to the eukaryotic cytochrome c oxidase. These oxidases catalyze the reduction of oxygen to water at a heme-copper binuclear center. Fourier transform infrared (FTIR) spectroscopy has been used to examine the heme-copper-containing respiratory oxidases of Rhodobacter sphaeroides Ga. This technique monitors the stretching frequency of CO bound at the oxygen binding site and can be used to characterize the oxidases in situ with membrane preparations. Oxidases that have a heme-copper binuclear center are recognizable by FTIR spectroscopy because the bound CO moves from the heme iron to the nearby copper upon photolysis at low temperature, where it exhibits a diagnostic spectrum. The FTIR spectra indicate that the binuclear center of the R. sphaeroides aa3-type cytochrome c oxidase is remarkably similar to that of the bovine mitochondrial oxidase. Upon deletion of the ctaD...
Archives of Biochemistry and Biophysics, 1999
The cyanobacteria Anacystis nidulans (Synechococcus sp. PCC6301), Synechocystis sp. PCC6803, Anabaena sp. PCC 7120, and Nostoc sp. PCC8009 were grown photoautotrophically under reduced oxygen tension in a medium with sulfate replaced by thiosulfate and nitrate replaced by ammonium as the Sand N-sources, respectively. In addition, Anabaena and Nostoc were grown under dinitrogen-fixing conditions in a medium free of combined nitrogen. Membranes were isolated from late-logarithmic cells (culture density corresponding to approximately 3 l packed cells per milliliter); cytoplasmic and thylakoid membranes were separated and purified according to established procedures. Acid-labile hemes were extracted from the membranes and subjected to reversed-phase highperformance liquid chromatography. Separated hemes were analyzed spectroscopically and identified by comparison with authentic standards. In addition to hemes B, A, and O, the latter of which was induced under semianaerobic conditions only, substitution of thiosulfate and ammonium for the oxy-anions sulfate and nitrate led to the appearance of spectrally discernible heme D in the membranes and extracts therefrom. However, spectroscopic and kinetic investigation of the membrane-bound heme D rather disproved any reaction with oxygen or carbon monoxide. Kinetic measurements performed with the membrane-bound respiratory oxidase gave evidence for only two kinetically competent terminal oxidases, a 3 and o 3 , both apparently associated with a single type of apoprotein, viz. subunit I of the known cyanobacterial aa 3
Critical structural role of R481 in cytochrome c oxidase from Rhodobacter sphaeroides
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2009
The R481 residue in cytochrome c oxidase from Rhodobacter sphaeroides forms hydrogen bonds with the propionate groups of both heme a and heme a 3. It has been postulated that R481 is the proton loading site in the proton exit pathway essential for proton translocation. A recent functional study showed that the mutations of R481 to His, Leu and Gln cause the reduction of the activity to ∼ 5-18% of the native level, and the absence of proton pumping in R481Q but retention of ∼ 40% efficiency in R481H and R481L (H.J. Lee, L. Öjemyr, A. Vakkasoglu, P. Brzezinski and R. B. Gennis, manuscript submitted). To decipher the molecular mechanism underlying the perturbed functionalities, we have used resonance Raman spectroscopy to examine the structural properties of the three mutants. The data show that the frequencies of the formyl Cf O stretching modes of both the heme a and a 3 in the mutants are characteristic of formyl groups exposed to an aqueous environment, indicating that the mutations disrupt the native H-bonding interaction between the formyl group of heme a and R52, as well as the hydrophobic environment surrounding the formyl group of heme a 3. In addition to the change in the environments of heme a and a 3 , the Raman data show that the mutations induce a partial conversion of the heme a 3 from a high-spin to a low-spin state, suggesting that the mutations are associated with the rearrangement of the Cu B-heme a 3 binuclear center. The Raman results reported here demonstrate that R481 plays a critical role in supporting efficient proton pumping, by holding the heme groups in a proper environment.
Journal of Biological Chemistry, 2006
Cytochrome c peroxidases (CCP) play a key role in cellular detoxification by catalyzing the reduction of hydrogen peroxide to water. The di-heme CCP from Rhodobacter capsulatus is the fastest enzyme (1060 s ؊1), when tested with its physiological cytochrome c substrate, among all di-heme CCPs characterized to date and has, therefore, been an attractive target to investigate structure-function relationships for this family of enzymes. Here, we combine for the first time structural studies with site-directed mutagenesis and spectroscopic studies of the mutant enzymes to investigate the roles of amino acid residues that have previously been suggested to be important for activity. The crystal structure of R. capsulatus at 2.7 Å in the fully oxidized state confirms the overall molecular scaffold seen in other di-heme CCPs but further reveals that a segment of about 10 amino acids near the peroxide binding site is disordered in all four molecules in the asymmetric unit of the crystal. Structural and sequence comparisons with other structurally characterized CCPs suggest that flexibility in this part of the molecular scaffold is an inherent molecular property of the R. capsulatus CCP and of CCPs in general and that it correlates with the levels of activity seen in CCPs characterized, thus, far. Mutagenesis studies support the spin switch model and the roles that Met-118, Glu-117, and Trp-97 play in this model. Our results help to clarify a number of aspects of the debate on structure-function relationships in this family of bacterial CCPs and set the stage for future studies. Cytochrome c peroxidases (CCP) 3 are found in yeast and bacteria and likely function to protect the organism against the accumulation of toxic peroxides. Cytochrome c peroxidase from Rhodobacter capsulatus (Rc) is a bacterial CCP (BCCP) that is located in the periplasm (1, 2). It binds two heme c groups covalently and utilizes the two co-factors as platforms to catalyze the two-electron reduction of hydrogen peroxide to water by a one-electron donor such as ferrocytochrome c. 2 Cyt c ͑Fe 2 ϩ ͒ ϩ H 2 O 2 ϩ 2 H ϩ 3 2 Cyt c ͑Fe 3 ϩ ͒ ϩ 2 H 2 O (Eq. 1) * This project has been supported by the Fund for Scientific Research-Flanders Contract G.0330.03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The atomic coordinates and structure factors (code 1ZZH) have been deposited in the Protein Data Bank,