Site-directed mutagenesis of five conserved residues of subunit i of the cytochrome cbb3 oxidase in Rhodobacter capsulatus

Cytochrome cbb(3) oxidase is a member of the heme-copper oxidase superfamily that catalyses the reduction of molecular oxygen to the water and conserves the liberated energy in the form of a proton gradient. Comparison of the amino acid sequences of subunit I from different classes of heme-copper oxidases showed that transmembrane helix VIII and the loop between transmembrane helices IX and X contain five highly conserved polar residues; Ser333, Ser340, Thr350, Asn390 and Thr394. To determine the relationship between these conserved amino acids and the activity and assembly of the cbb(3) oxidase in Rhodobacter capsulatus, each of these five conserved amino acids was substituted for alanine by site-directed mutagenesis. The effects of these mutations on catalytic activity were determined using a NADI plate assay and by measurements of the rate of oxygen consumption. The consequence of these mutations for the structural integrity of the cbb(3) oxidase was determined by SDS-PAGE analysis of chromatophore membranes followed by TMBZ staining. The results indicate that the Asn390Ala mutation led to a complete loss of enzyme activity and that the Ser333Ala mutation decreased the activity significantly. The remaining mutants cause a partial loss of catalytic activity. All of the mutant enzymes, except Asn390Ala, were apparently correctly assembled and stable in the membrane of the R. capsulatus.     

Biogenesis of cbb3-type cytochrome c oxidase in Rhodobacter capsulatus

The cbb₃-type cytochrome c oxidases (cbb₃-Cox) constitute the second most abundant cytochrome c oxidase (Cox) group after the mitochondrial-like aa₃-type Cox. They are present in bacteria only, and are considered to represent a primordial innovation in the domain of Eubacteria due to their phylogenetic distribution and their similarity to nitric oxide (NO) reductases. They are crucial for the onset of many anaerobic biological processes, such as anoxygenic photosynthesis or nitrogen fixation. In addition, they are prevalent in many pathogenic bacteria, and important for colonizing low oxygen tissues. Studies related to cbb₃-Cox provide a fascinating paradigm for the biogenesis of sophisticated oligomeric membrane proteins. Complex subunit maturation and assembly machineries, producing the c-type cytochromes and the binuclear heme b₃-Cu_B center, have to be coordinated precisely both temporally and spatially to yield a functional cbb₃-Cox enzyme. In this review we summarize our current knowledge on the structure, regulation and assembly of cbb₃-Cox, and provide a highly tentative model for cbb₃-Cox assembly and formation of its heme b₃-Cu_B binuclear center. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

Multi-step assembly pathway of the cbb3-type cytochrome c oxidase complex

The cbb3-type cytochrome c oxidases as members of the heme-copper oxidase superfamily are involved in microaerobic respiration in both pathogenic and non-pathogenic proteobacteria. The biogenesis of these multisubunit enzymes, encoded by the ccoNOQP operon, depends on the ccoGHIS gene products, which are proposed to be specifically required for co-factor insertion and maturation of cbb3-type cytochrome c oxidases. Here, the assembly of the cbb3-type cytochrome c oxidase from the facultative photosynthetic model organism Rhodobacter capsulatus was investigated using blue-native polyacrylamide gel electrophoresis. This process involves the formation of a stable but inactive 210 kDa sub-complex consisting of the subunits CcoNOQ and the assembly proteins CcoH and CcoS. By recruiting monomeric CcoP, this sub-complex is converted into an active 230 kDa CcoNOQP complex. Formation of these complexes and the stability of the monomeric CcoP are impaired drastically upon deletion of ccoGHIS. In a ccoI deletion strain, the 230 kDa complex was absent, although monomeric CcoP was still detectable. In contrast, neither of the complexes nor the monomeric CcoP was found in a ccoH deletion strain. In the absence of CcoS, the 230 kDa complex was assembled. However, it exhibited no enzymatic activity, suggesting that CcoS might be involved in a late step of biogenesis. Based on these data, we propose that CcoN, CcoO and CcoQ assemble first into an inactive 210 kDa sub-complex, which is stabilized via its interactions with CcoH and CcoS. Binding of CcoP, and probably subsequent dissociation of CcoH and CcoS, then generates the active 230 kDa complex. The insertion of the heme cofactors into the c-type cytochromes CcoP and CcoO precedes sub-complex formation, while the cofactor insertion into CcoN could occur either before or after the 210 kDa sub-complex formation during the assembly of the cbb3-type cytochrome c oxidase.     

Oxygen adaptation. The role of the CcoQ subunit of the cbb3 cytochrome c oxidase of Rhodobacter sphaeroides 2.4.1

The cbb(3) cytochrome c oxidase of Rhodobacter sphaeroides consists of four nonidentical subunits. Three subunits (CcoN, CcoO, and CcoP) comprise the catalytic "core" complex required for the reduction of O(2) and the oxidation of a c-type cytochrome. On the other hand, the functional role of subunit IV (CcoQ) of the cbb(3) oxidase was not obvious, although we previously suggested that it is involved in the signal transduction pathway controlling photosynthesis gene expression (Oh, J. I., and Kaplan, S. (1999) Biochemistry 38, 2688-2696). Here we go on to demonstrate that subunit IV protects the core complex, in the presence of O(2), from proteolytic degradation by a serine metalloprotease. In the absence of CcoQ, we suggest that the presence of O(2) leads to the loss of heme from the core complex, which destabilizes the cbb(3) oxidase into a "degradable" form, perhaps by altering its conformation. Under aerobic conditions the absence of CcoQ appears to affect the CcoP subunit most severely. It was further demonstrated, using a series of COOH-terminal deletion derivatives of CcoQ, that the minimum length of CcoQ required for stabilization of the core complex under aerobic conditions is the amino-terminal approximately 48-50 amino acids.     

Biogenesis of cbb(3)-type cytochrome c oxidase in Rhodobacter capsulatus

The cbb(3)-type cytochrome c oxidases (cbb(3)-Cox) constitute the second most abundant cytochrome c oxidase (Cox) group after the mitochondrial-like aa(3)-type Cox. They are present in bacteria only, and are considered to represent a primordial innovation in the domain of Eubacteria due to their phylogenetic distribution and their similarity to nitric oxide (NO) reductases. They are crucial for the onset of many anaerobic biological processes, such as anoxygenic photosynthesis or nitrogen fixation. In addition, they are prevalent in many pathogenic bacteria, and important for colonizing low oxygen tissues. Studies related to cbb(3)-Cox provide a fascinating paradigm for the biogenesis of sophisticated oligomeric membrane proteins. Complex subunit maturation and assembly machineries, producing the c-type cytochromes and the binuclear heme b(3)-Cu(B) center, have to be coordinated precisely both temporally and spatially to yield a functional cbb(3)-Cox enzyme. In this review we summarize our current knowledge on the structure, regulation and assembly of cbb(3)-Cox, and provide a highly tentative model for cbb(3)-Cox assembly and formation of its heme b(3)-Cu(B) binuclear center. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

Dynamic water networks in cytochrome cbb3 oxidase

Heme-copper oxidases (HCOs) are terminal electron acceptors in aerobic respiration. They catalyze the reduction of molecular oxygen to water with concurrent pumping of protons across the mitochondrial and bacterial membranes. Protons required for oxygen reduction chemistry and pumping are transferred through proton uptake channels. Recently, the crystal structure of the first C-type member of the HCO superfamily was resolved [Buschmann et al. Science 329 (2010) 327–330], but crystallographic water molecules could not be identified. Here we have used molecular dynamics (MD) simulations, continuum electrostatic approaches, and quantum chemical cluster calculations to identify proton transfer pathways in cytochrome cbb₃. In MD simulations we observe formation of stable water chains that connect the highly conserved Glu323 residue on the proximal side of heme b₃ both with the N- and the P-sides of the membrane. We propose that such pathways could be utilized for redox-coupled proton pumping in the C-type oxidases. Electrostatics and quantum chemical calculations suggest an increased proton affinity of Glu323 upon reduction of high-spin heme b₃. Protonation of Glu323 provides a mechanism to tune the redox potential of heme b₃ with possible implications for proton pumping.     

The identification of histidine ligands to cytochrome a in cytochrome c oxidase

A histidine auxotroph of Saccharomyces cerevisiae has been used to metabolically incorporate [1,3-15N2] histidine into yeast cytochrome c oxidase. Electron nuclear double resonance (ENDOR) spectroscopy of cytochrome a in the [15N]histidine-substituted enzyme reveals an ENDOR signal which can be assigned to hyperfine coupling of a histidine 15N with the low-spin heme, thereby unambiguously identifying histidine as an axial ligand to this cytochrome. Comparison of this result with similar ENDOR data obtained on two 15N-substituted bisimidazole model compounds, metmyoglobin-[15N]imidazole and bis[15N]imidazole tetraphenyl porphyrin, provides strong evidence for bisimidazole coordination in cytochrome a.     

Proton translocation by cytochrome c oxidase can take place without the conserved glutamic acid in subunit I

A glutamic acid residue in subunit I of the heme-copper oxidases is highly conserved and has been directly implicated in the O(2) reduction and proton-pumping mechanisms of these respiratory enzymes. Its mutation to residues other than aspartic acid dramatically inhibits activity, and proton translocation is lost. However, this glutamic acid is replaced by a nonacidic residue in some structurally distant members of the heme-copper oxidases, which have a tyrosine residue in the vicinity. Here, using cytochrome c oxidase from Paracoccus denitrificans, we show that replacement of the glutamic acid and a conserved glycine nearby lowers the catalytic activity to <0.1% of the wild-type value. But if, in addition, a phenylalanine that lies close in the structure is changed to tyrosine, the activity rises more than 100-fold and proton translocation is restored. Molecular dynamics simulations suggest that the tyrosine can support a transient array of water molecules that may be essential for proton transfer in the heme-copper oxidases. Surprisingly, the glutamic acid is thus not indispensable, which puts important constraints on the catalytic mechanism of these enzymes.     

Cloning of Paracoccus cytochrome c oxidase subunit II

Cytochrome c oxidase from Paracoccus denitrificans is composed of two subunits, yet is active in both electron transport and proton translocation. A cloning approach and immunologic screening protocol is described for the isolation of the subunit II gene expressed in E. coli. DNA sequencing should establish the extent of homology to eukaryotic oxidase.     

Effect of single-site charge-reversal mutations on the catalytic properties of yeast cytochrome c peroxidase: mutations near the high-affinity cytochrome c binding site

Fifteen single-site charge-reversal mutations of yeast cytochrome c peroxidase (CcP) have been constructed to determine the effect of localized charge on the catalytic properties of the enzyme. The mutations are located on the front face of CcP, near the cytochrome c binding site identified in the crystallographic structure of the yeast cytochrome c-CcP complex [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755]. The mutants are characterized by absorption spectroscopy and hydrogen peroxide reactivity at both pH 6.0 and 7.5 and by steady-state kinetic studies using recombinant yeast iso-1-ferrocytochrome c(C102T) as a substrate at pH 7.5. Some of the charge-reversal mutations cause detectable changes in the absorption spectrum, especially at pH 7.5, reflecting changes in the equilibrium between penta- and hexacoordinate heme species in the enzyme. An increase in the amount of hexacoordinate heme in the mutant enzymes correlates with an increase in the fraction of enzyme that does not react with hydrogen peroxide. Steady-state velocity measurements indicate that five of the 15 mutations cause large increases in the Michaelis constant (R31E, D34K, D37K, E118K, and E290K). These data support the hypothesis that the cytochrome c-CcP complex observed in the crystal is the dominant catalytically active complex in solution.     

Time dependence of the catalytic intermediates in cytochrome c oxidase

Cytochrome c oxidase, the terminal enzyme in the electron transfer chain, catalyzes the reduction of oxygen to water in a multiple step process by utilizing four electrons from cytochrome c. To study the reaction mechanism, the resonance Raman spectra of the intermediate states were measured during single turnover of the enzyme after catalytic initiation by photolysis of CO from the fully reduced CO-bound enzyme. By measuring the change in intensity of lines associated with heme a, the electron transfer steps were determined and found to be biphasic with apparent rate constants of approximately 40 x 10(3) s(-1) and approximately 1 x 10(3) s(-1). The time dependence for the oxidation of heme a and for the measured formation and decay of the oxy, the ferryl ("F"), and the hydroxy intermediates could be simulated by a simple reaction scheme. In this scheme, the presence of the "peroxy" ("P") intermediate does not build up a sufficient population to be detected because its decay rate is too fast in buffered H(2)O at neutral pH. A comparison of the change in the spin equilibrium with the formation of the hydroxy intermediate demonstrates that this intermediate is high spin. We also confirm the presence of an oxygen isotope-sensitive line at 355 cm(-1), detectable in the spectrum from 130 to 980 micros, coincident with the presence of the F intermediate.     

Subunit function in eukaryote cytochrome c oxidase. A mutation in the nuclear-coded subunit IV allows assembly but alters the function and stability of yeast cytochrome c oxidase.

Strains of the yeast Saccharomyces cerevisiae disrupted in YCOX4, the nuclear gene encoding cytochrome c oxidase subunit IV, do not assemble a functional or spectrally visible oxidase. We report the characterization of a yeast strain, RM1, expressing a mutated YCOX4 gene which is temperature sensitive for respiration at 37 degrees C, but incorporates cytochrome aa3 over all growth temperatures. The mutant enzyme is less stable than the wild type, with subunit IV readily proteolyzed without gross denaturation of the complex but with a concomitant loss of oxidase activity. When grown fermentatively at 37 degrees C, cytochrome c oxidase from the mutant strain had a turnover number of less than 3% of the normal complex, while Km values and subunit levels were comparable to normal. Thus alterations in subunit IV can perturb the enzyme structure and alter its catalytic rate, implying a role for this subunit in cytochrome c oxidase function as distinct from assembly.     

Properties of the detergent solubilised cytochrome c oxidase (cytochrome cbb(3)) purified from Pseudomonas stutzeri.

Cytochrome cbb(3) is a cytochrome c-oxidising isoenzyme that belongs to the superfamily of respiratory haem/copper oxidases. We have developed a purification method yielding large amounts of pure cbb(3) complex from the soil bacterium Pseudomonas stutzeri. This cytochrome cbb(3) complex consists of three subunits (ccoNOP) in a 1:1:1 stoichiometry and contains two b-type and three c-type haems. The protein complex behaves as a monomer with an overall molecular weight of 114.0+/-8.9 kDa and a s(0)(20,w) value of 8.9+/-0.3 S as determined by analytical ultracentrifugation. Crystals diffracting to 5.0 A resolution have been grown by the vapour diffusion sitting drop method to an average size of 0.1 x 0.1 x 0.3 mm. This is the first crystallisation report of a (cbb(3))-type oxidase.     

The subunit structure of the cytochrome c oxidase complex.

The subunit structure of the cytochrome c oxidase complex has been obtained for three preparations each isolated by a different detergent procedure. Six polypeptides were present in all samples with the following molecular weights: subunits I, 36000; II, 22500, III, 17100; IV, 12500; V, 9700; and VI, 5300. These subunits have been purified by gel filtration in sodium dodecyl sulfate or in 6 M guanidine hydrochloride and their amino acid compositions have been determined. Subunit I is hydrophobic in character with a polarity of 35.7%. Subunits II through VI are more hydrophilic with polarities of 45.5, 48.6, 47.8, 49.7, and 53.7%, respectively.     

Evolution of the primate cytochrome c oxidase subunit II gene

We examined the nucleotide and amino acid sequence variation of the cytochrome c oxidase subunit II (COII) gene from 25 primates (4 hominoids, 8 Old World monkeys, 2 New World monkeys, 2 tarsiers, 7 lemuriforms, 2 lorisiforms). Marginal support was found for three phylogenetic conclusions: (1) sister-group relationship between tarsiers and a monkey/ape clade, (2) placement of the aye-aye (Daubentonia) sister to all other strepsirhine primates, and (3) rejection of a sister-group relationship of dwarf lemurs (i.e., Cheirogaleus) with lorisiform primates. Stronger support was found for a sister-group relationship between the ring-tail lemur (Lemur catta) and the gentle lemurs (Hapalemur). In congruence with previous studies on COII, we found that the monkeys and apes have undergone a nearly two-fold increase in the rate of amino acid replacement relative to other primates. Although functionally important amino acids are generally conserved among all primates, the acceleration in amino acid replacements in higher primates is associated with increased variation in the amino terminal end of the protein. Additionally, the replacement of two carboxyl-bearing residues (glutamate and aspartate) at positions 114 and 115 may provide a partial explanation for the poor enzyme kinetics in cross-reactions between the cytochromes c and cytochrome c oxidases of higher primates and other mammals. Correspondence to: R.L. Honeycutt     

Effect of subunit III removal on control of cytochrome c oxidase activity by pH

Studies were undertaken to assess the postulated involvement of subunit III in the proton-linked functions of cytochrome c oxidase. The effect of pH on the steady-state kinetic [corrected] parameters of subunit III containing and subunit III depleted cytochrome oxidase was determined by using beef heart and rat liver enzymes reconstituted into phospholipid vesicles. The TNmax and Km values for the III-containing enzyme increase with decreasing pH in a manner quantitatively similar to that reported by Thornstrom et al. [(1984) Chem. Scr. 24, 230-235], giving three apparent pKa values of less than 5.0, 6.2, and 7.8. The maximal activities of the subunit III depleted enzymes (beef heart and rat liver) show a similar dependence on pH, but the Km values are consistently higher than those of the III-containing enzyme, an effect that is accentuated at low pH. The pH dependence of TNmax/Km for both forms of the enzyme (+/- subunit III) indicates that protonation of a group with an apparent pKa of 5.7 lowers the affinity for substrate (cytochrome c) independently of a continued increase in maximal velocity. N,N'-Dicyclohexylcarbodiimide (DCCD) decreases the pH responsiveness of the electron-transfer activity to the same extent in both III-containing and III-depleted enzymes, indicating that this effect is mediated by a peptide other than subunit III. Control of intramolecular electron transfer by a transmembrane pH gradient (or alkaline intravesicular pH) is shown to occur in cytochrome oxidase vesicles with cytochrome c as the electron donor, in agreement with results of Moroney et al. [(1984) Biochemistry 23, 4991-4997] using hexaammineruthenium(II) as the reductant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of mutation of the conserved glutamic acid-286 in subunit I of cytochrome c oxidase from Rhodobacter sphaeroides

We have studied the effects of mutations, E286Q and E286D, of the conserved glutamate in subunit I of cytochrome c oxidase from Rhodobacter sphaeroides with a view to evaluating the role of this residue in redox-linked proton translocation. The mutation E286D did not have any dramatic effects on enzyme properties and retained 50% of wild-type catalytic activity. For E286Q a fraction of the binuclear center was trapped in an unreactive, spectrally distinct form which is most likely due to misfolded protein, but the majority of E286Q reacted normally with formate and cyanide in the oxidized state, and with carbon monoxide and cyanide in the dithionite-reduced form. The mutation also had little effect on the pH-dependent redox properties of haem a in the reactive fraction. However, formation of the P state from oxidized enzyme with hydrogen peroxide or by aerobic incubation with carbon monoxide was inhibited. In particular, only an F-type product was obtained, at less than 25% yield, in the reaction with hydrogen peroxide. The aerobic steady state in the presence of ferrous cytochrome c was characterized by essentially fully reduced haem a and ferric haem a3, suggesting that the mutation hinders electron transfer from haem a to the binuclear center. Under these conditions or after reoxidation, on a seconds time scale, of haem a3 following anaerobiosis, there was no indication of accumulation of significant amounts of P state. We propose that the glutamate is implicated in several steps in the catalytic cycle, O --> R, P --> F, and, possibly, F --> O. The results are discussed in relation to the "glutamate trap" model for proton translocation.     

Dynamic water networks in cytochrome cbb(3) oxidase

Heme-copper oxidases (HCOs) are terminal electron acceptors in aerobic respiration. They catalyze the reduction of molecular oxygen to water with concurrent pumping of protons across the mitochondrial and bacterial membranes. Protons required for oxygen reduction chemistry and pumping are transferred through proton uptake channels. Recently, the crystal structure of the first C-type member of the HCO superfamily was resolved [Buschmann et al. Science 329 (2010) 327-330], but crystallographic water molecules could not be identified. Here we have used molecular dynamics (MD) simulations, continuum electrostatic approaches, and quantum chemical cluster calculations to identify proton transfer pathways in cytochrome cbb(3). In MD simulations we observe formation of stable water chains that connect the highly conserved Glu323 residue on the proximal side of heme b(3) both with the N- and the P-sides of the membrane. We propose that such pathways could be utilized for redox-coupled proton pumping in the C-type oxidases. Electrostatics and quantum chemical calculations suggest an increased proton affinity of Glu323 upon reduction of high-spin heme b(3). Protonation of Glu323 provides a mechanism to tune the redox potential of heme b(3) with possible implications for proton pumping.