Mammalian cytochrome c oxidase, a molecular monster subdued.
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Animals↗
Biomedical subjects
S Ferguson-Miller
Publications and source records attributed to S Ferguson-Miller.
A ligand-exchange mechanism of proton pumping involving tyrosine-422 of subunit I of cytochrome oxidase is ruled out.
The molecular mechanism by which proton pumping is coupled to electron transfer in cytochrome c oxidase has not yet been determined. However, several models of this process have been proposed which are based on changes occurring in the vicinity of the redox centers of the enzyme. Recently, a model was described in which a well-conserved tyrosine residue in subunit I (Y422) was proposed to undergo ligand exchange with the histidine ligand (H419) of the high-spin heme a3 during the catalytic cycle, allowing both residues to serve as part of a proton transporting system. Site-directed mutants of Y422 have been constructed in the aa3-type cytochrome c oxidase of Rhodobacter sphaeroides to test this hypothesis (Y422A, Y422F). The results demonstrate that Y422 is not an essential residue in the electron transfer and proton pumping mechanisms of cytochrome c oxidase. However, the results support the predicted proximity of Y422 to heme a3, as now confirmed by crystal structure. In addition, it is shown that the pH-dependent reversed electron transfer between heme a and heme a3 is normal in the Y422F mutant. Hence, these data also demonstrate that Y422 is not the residue previously postulated to interact electrostatically with heme a3, nor is it responsible for the unique EPR characteristics of heme a in this bacterial oxidase.
Base Sequence↗
Protein structure: proton-pumping oxidases.
The crystal structures of two cytochrome c oxidases, one bacterial and one mammalian, offer insights into their roles in oxygen chemistry and as proton pumps.
Animals↗
Structure of cytochrome c oxidase, energy generator of aerobic life.
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Aerobiosis↗
Two conformations of the catalytic site in the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides.
Resonance Raman spectra of the carbon monoxy derivative of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides show two distinct Fe-CO stretching modes (519 and 493 cm-1) at room temperature. The frequency of the mode at 519 cm-1 coincides with that of other terminal oxidases at neutral pH. Two C-O stretching modes, one at 1966 cm-1 and one at 1955 cm-1, are also found. The splitting of the C-O stretching mode is consistent with the FTIR spectra of cytochrome c oxidases at cryogenic temperatures in which two different conformations (alpha and beta) of the catalytic site of the enzyme are present. The splitting of both the Fe-CO and C-O stretching modes under our conditions indicates that these two forms of the enzyme are also present at room temperature, and with the additional information on the Fe-CO modes provided here, a structural origin for the two forms may be postulated. The alpha-form has the same general structure of the active site as mammalian oxidase, a structure in which the copper atom that is the part of the Fe-CuB binuclear site interacts strongly with the bound CO. We postulate that the copper atom exerts a strong polar or steric effect on the heme-bound CO, resulting in either compression of the Fe-CO bond or distortion of the Fe-CO moiety.(ABSTRACT TRUNCATED AT 250 WORDS)
Binding Sites↗
Analysis of site-directed mutants locates a non-redox-active metal near the active site of cytochrome c oxidase of Rhodobacter sphaeroides.
Substoichiometric amounts of Mn are bound by the aa3-type cytochrome c oxidase of Rhodobacter sphaeroides and appear in the EPR spectrum of the purified enzyme as signals that overlay those of CuA in the g = 2.0 region. The Mn is tightly bound and not removed by a high degree of purification or by washing with 50 mM EDTA. The amount of bound Mn varies with the ratio of Mg to Mn in the growth medium. Oxidase containing no EPR-detectable Mn can be prepared from cells grown in low Mn/Mg, while high Mn/Mg in the growth medium gives rise to near stoichiometric levels (0.7 mol/mol of aa3). Incubation of purified Mn-deficient oxidase with 1 mM Mn does not allow incorporation into the tight binding site, indicating that this site is not accessible in the assembled protein. When bound Mn is depleted by growth in high Mg, there is no change in electron transfer activity, suggesting that Mg may substituted for Mn and maintain protein structure. Analysis of site-directed mutants in an extramembrane loop close to the active site of cytochrome oxidase identifies His-411 and Asp-412 of subunit I as probable ligands of the Mn. Mutation of either residue leads to lower activity and loss of Mn binding, even in cells grown in elevated concentrations of Mn. Since Mn binding correlates with the [Mn] to [Mg] ratio in the culture medium, we propose that Mn competes for the site that normally binds a stoichiometric Mg ion in aa3-type cytochrome c oxidases.
Amino Acid Sequence↗
A continuous wave and pulsed EPR characterization of the Mn2+ binding site in Rhodobacter sphaeroides cytochrome c oxidase.
The ligation environment of the tightly bound Mn2+ in cytochrome c oxidase from Rhodobacter sphaeroides has been characterized by electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM). The EPR data show that the Mn2+ is six-coordinate and located in a highly symmetric binding site. Analyses of X- and Q-band EPR spectra show that the zero field splitting parameter D is 115 +/- 25 G (0.0107 +/- 0.0023 cm-1) in the fully oxidized enzyme and 125 +/- 15 G (0.0117 +/- 0.0014 cm-1) in the fully reduced enzyme. For both redox forms of the enzyme the value of E is < or = 25 G (0.0023 cm-1). By comparison with crystal structures of Mn2+ binding proteins, the structural changes at the Mn2+ binding site upon redox state change of the enzyme are estimated to be < or = 0.2 A in ligand bond lengths and < or = 10 degrees in bond angle. This analysis indicates that little modification occurs at the Mn2+ site upon redox change at the other metal centers. Considering the proximity of the Mn2+ site to heme a and heme a3-CuB [Hosler, J. P., Espe, M. P., Zhen, Y., Babcock, G. T., & Ferguson-Miller, S. (1995) Biochemistry 34, 7586-7592], we interpret these results to imply also that there is no large protein conformational change near the heme a and heme a3-CuB sites upon a change in their redox states. Multifrequency 3-pulse ESEEM results provide direct evidence for a nitrogen ligand to the Mn2+, which is assigned to a histidine by comparison with ESEEM studies of Mn(2+)-bound lectins [McCracken, J., Peisach, J., Bhattacharyya, L., & Brewer, F. (1991) Biochemistry 30, 4486-4491] and specifically to His-411 in subunit 1 on the basis of mutagenesis studies (Hosler et al., 1995). From these results a partial model of the Mn2+ binding site has been constructed.
Binding Sites↗
Possible proton relay pathways in cytochrome c oxidase.
As the final electron acceptor in the respiratory chain of eukaryotic and many prokaryotic organisms, cytochrome c oxidase (EC 1.9.3.1) catalyzes the reduction of oxygen to water and generates a proton gradient. To test for proton pathways through the oxidase, site-directed mutagenesis was applied to subunit I of the Rhodobacter sphaeroides enzyme. Mutants were characterized in three highly conserved regions of the peptide, comprising possible proton loading, unloading, and transfer sites: an interior loop between helices II and III (Asp132Asn/Ala), an exterior loop between helices IX and X (His411Ala, Asp412Asn, Thr413Asn, Tyr414Phe), and the predicted transmembrane helix VIII (Thr352Ala, Pro358Ala, Thr359Ala, Lys362Met). Most of the mutants had lower activity than wild type, but only mutants at residue 132 lost proton pumping while retaining electron transfer activity. Although electron transfer was substantially inhibited, no major structural alteration appears to have occurred in D132 mutants, since resonance Raman and visible absorbance spectra were normal. However, lower CO binding (70-85% of wild type) suggests some minor change to the binuclear center. In addition, the activity of the reconstituted Asp132 mutants was inhibited rather than stimulated by ionophores or uncoupler. The inhibition was not observed with the purified enzyme and a direct pH effect was ruled out, suggesting an altered response to the electrical or pH gradient. The results support an important role for the conserved II-III loop in the proton pumping process and are consistent with the possibility of involvement of residues in helix VIII and the IX-X loop.
Amino Acid Sequence↗
Identity of the axial ligand of the high-spin heme in cytochrome oxidase: spectroscopic characterization of mutants in the bo-type oxidase of Escherichia coli and the aa3-type oxidase of Rhodobacter sphaeroides.
Prokaryotic and eukaryotic cytochrome c oxidases and several bacterial ubiquinol oxidases compose a superfamily of heme-copper oxidases. These enzymes are terminal components of aerobic respiratory chains, the principal energy-generating systems of aerobic organisms. Two such heme-copper oxidases are the aa3-type cytochrome c oxidase of Rhodobacter sphaeroides and the bo-type ubiquinol oxidase of Escherichia coli. These enzymes catalyze the reduction of oxygen to water at a heme-copper binuclear center. Energy conservation is accomplished by coupling electron transfer through the metals of the oxidases to proton translocation across the cellular membrane. The Rb. sphaeroides and E. coli enzymes have previously been utilized in site-directed mutagenesis studies which identified two histidines which bind the low-spin heme (heme a), as well as additional histidine residues which are probable ligands for copper (CuB). However, the histidine that binds the heme of the binuclear center (heme a3) could not be unequivocally identified between two residues (His284 and His419). Additional characterization by Fourier transform infrared spectroscopy of the CO-bound forms of the E. coli enzyme in which His284 is replaced by glycine or leucine demonstrates that these mutations cause only subtle changes to CO bound to the heme of the binuclear center. Resonance Raman spectroscopy of the Rb. sphaeroides enzyme in which His284 is replaced by alanine shows that the iron-histidine stretching mode of heme a3 is maintained, in contrast with the loss of this mode in mutants at His419. These results demonstrate that His284 is not the heme a3 ligand.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Insight into the active-site structure and function of cytochrome oxidase by analysis of site-directed mutants of bacterial cytochrome aa3 and cytochrome bo.
Cytochrome aa3 of Rhodobacter sphaeroides and cytochrome bo of E. coli are useful models of the more complex cytochrome c 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 heme a3 (or o)-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.
Amino Acid Sequence↗
Cytochrome aa3 of Rhodobacter sphaeroides as a model for mitochondrial cytochrome c oxidase. Purification, kinetics, proton pumping, and spectral analysis.
Aerobically grown Rhodobacter sphaeroides synthesizes a respiratory chain similar to that of eukaryotes. We describe the purification of the aa3-type cytochrome c oxidase of Rb. sphaeroides as a highly active (Vmax > or = 1800 s-1), three-subunit enzyme from isolated, washed cytoplasmic membranes by hydroxylapatite chromatography and anion exchange fast protein liquid chromatography. The purified oxidase exhibits biphasic kinetics of oxidation of mammalian cytochrome c, similar to mitochondrial oxidases, and pumps protons efficiently (H+/e- = 0.7) following reconstitution into phospholipid vesicles. A membrane-bound cytochrome c is associated with the aa3-type oxidase in situ, but is removed during purification. The EPR spectra of the Rb. sphaeroides enzyme suggest the presence of a strong hydrogen bond to one or both of the histidine ligands of heme a. In other respects, optical, EPR, and resonance Raman analyses of the metal centers and their protein environments demonstrate a close correspondence between the bacterial enzyme and the structurally more complex bovine cytochrome c oxidase. The results establish this bacterial oxidase as an excellent model system for the mammalian enzyme and provide the basis for site-directed mutational analysis of its energy transducing function.
Animals↗
Cytochrome aa3 of Rhodobacter sphaeroides as a model for mitochondrial cytochrome c oxidase. The coxII/coxIII operon codes for structural and assembly proteins homologous to those in yeast.
The coxII/coxIII operon of Rhodobacter sphaeroides cytochrome c oxidase has been sequenced and characterized by insertional inactivation/complementation analysis. The organization of the genes in this locus (coxII.orf1.orf3.coxIII) is the same as that of the equivalent operon of Paracoccus denitrificans (ctaC.ctaB.ctaG.ctaE), but unlike that of other bacteria whose cytochrome oxidase genes have been characterized so far. The predicted amino acid sequence homology with eukaryotic oxidases is also higher for Rb. sphaeroides (and P. denitrificans) than for other bacterial versions of the enzyme. The inactivation of coxII results in loss of the characteristic cytochrome oxidase spectrum from membranes of the mutant strain. Full recovery requires introduction into the bacterium of the complete operon containing coxII.orf1.orf3.coxIII; partial complementation yielding a spectrally altered enzyme is achieved with a plasmid containing coxII or coxII.orf1.orf3. These results indicate that the peptides ORF1, ORF3, and COXIII are all required for assembly of native cytochrome c oxidase, suggesting an oxidase-specific assembly or chaperonin function for the ORFs in Rb. sphaeroides similar to that observed for the homologous gene products in yeast, COX10 and COX11.
Amino Acid Sequence↗
Interaction of cytochrome c with cytochrome c oxidase studied by monoclonal antibodies and a protein modifying reagent.
C/57 black mice were immunized with beef heart cytochrome c oxidase, generating 48 hybrid cell lines that secrete antibodies against the different subunits of the enzyme. Immunoblot analysis showed reactions with 7 of the 13 subunits. Among the monoclonal antibodies produced, only those to subunit II gave significant inhibition; these inhibited the enzyme activity completely and prevented cytochrome c binding to the enzyme. Epitope mapping studies indicate that a peptide including residues 200-227 reacts with the antibody, suggesting that the C-terminus of the protein is essential for the binding of this antibody. The carboxyl modifying reagent 1-ethyl-3-[3-(trimethylammonio)propyl]carbodiimide (ETC) was chosen to investigate further the relationship between antibody and cytochrome c binding domains. ETC caused 50% inhibition of the enzyme activity with a first-order time during the first 20 min; a slower reaction over 3 h resulted in 90% inhibition. Cytochrome c binding to the oxidase was inhibited to a similar extent as cytochrome c oxidation, and protection against both effects was afforded by the presence of cytochrome c during ETC modification. Anion-exchange of FPLC of the modified forms of cytochrome oxidase revealed extensive inhomogeneity, indicating random derivatization of a number of different carboxyls even during the first-order reaction, and precluding identification of carboxyl residues related to a specific phase of the reaction. Cytochrome c and the subunit II-specific antibody protected against radioactive labeling of subunit II by ETC in the presence of [14C]glycine ethyl ester, demonstrating that the antibody and cytochrome c occupy significant and overlapping areas on the subunit II surface.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Definition of the catalytic site of cytochrome c oxidase: specific ligands of heme a and the heme a3-CuB center.
The three-subunit aa3-type cytochrome c oxidase (EC 1.9.3.1) of Rhodobacter sphaeroides is structurally and functionally homologous to the more complex mitochondrial oxidase. The largest subunit, subunit I, is highly conserved and predicted to contain 12 transmembrane segments that provide all the ligands for three of the four metal centers: heme a, heme a3, and CuB. A variety of spectroscopic techniques identify these ligands as histidines. We have used site-directed mutagenesis to change all the conserved histidines within subunit I of cytochrome c oxidase from Rb. sphaeroides. Analysis of the membrane-bound and purified mutant proteins by optical absorption and resonance Raman spectroscopy indicates that His-102 and His-421 are the ligands of heme a, while His-284, His-333, His-334, and His-419 ligate the heme a3-CuB center. To satisfy this ligation assignment, helices II, VI, VII, and X, which contain these histidine residues, must be in close proximity. These data provide empirical evidence regarding the three-dimensional protein structure at the catalytic core of cytochrome c oxidase.
Amino Acid Sequence↗
The gene encoding cytochrome c oxidase subunit II from Rhodobacter sphaeroides; comparison of the deduced amino acid sequence with sequences of corresponding peptides from other species.
The gene (coxII) encoding subunit II of Rhodobacter sphaeroides cytochrome c oxidase (cytochrome aa3) has been isolated by screening a genomic DNA library in phage lambda with a probe derived from coxII of Paracoccus denitrificans. A 2-kb fragment containing coxII DNA was subcloned into the phage M13mp18 and the sequence determined. The 2-kb insert contains the entire coding region for coxII gene, including the ATG start codon and a TGA stop codon. The deduced amino acid (aa) sequence of subunit II of R. sphaeroides shows regions of substantial homology to the corresponding subunit of the bovine mitochondrial oxidase (63% overall) and P. denitrificans oxidase (68% overall). The postulated redox-active copper ion (CuA) binding site involving two Cys and two His residues (as well as an alternative Met residue) is conserved among these species, along with four invariant acidic aa residues (two Asp and two Glu) that may be involved in interactions with cytochrome c, and a region of aromatic residues (Tyr-Gln-Trp-Tyr-Trp-Gly-Tyr-Glu-Tyr) which is postulated to play a role in electron transfer. Hydropathy profile analysis suggests that while the bovine COXII secondary structure contains two transmembrane helices, the R. sphaeroides subunit II has a third such helix that may function as part of a signal sequence, as suggested for P. denitrificans.
Amino Acid Sequence↗
Conversion of a mitochondrial gene for mammalian cytochrome c oxidase subunit II into its universal codon equivalent and expression in vivo and in vitro.
To begin to assess the independent structural and functional characteristics of the mitochondrially encoded subunits of mammalian cytochrome c oxidase, we have converted the cloned mitochondrial gene for rat subunit II (coxII) into its universal codon equivalent (ucoxII) by oligonucleotide-directed, site-specific mutagenesis. This involved synthesizing 12 oligodeoxynucleotides to achieve the 13 ATA to ATG and the 5 TGA to TGG changes needed. To express ucoxII in Escherichia coli, we used a number of different expression vectors in which the promoters and ribosome-binding sequences of the messenger RNA were varied. While ucoxII alone was expressed at a low level, a striking increase in the level of expression resulted when the ucoxII gene was fused to other E. coli genes. The COXII peptide was identified by proteolytic digestion, partial sequencing, and reaction with specific antisera. A cro-beta-galactosidase-COXII fusion protein has been purified, characterized, and used to produce polyclonal antibodies to the COXII peptide. The ucoxII gene was also expressed in a cell-free translation system and in Xenopus oocytes, yielding a nondenatured, membrane-associated peptide with the same apparent molecular weight as authentic subunit II. In oocytes and in a reticulocyte lysate in vitro system supplemented with microsomal membranes, the protein is glycosylated and coisolates with the washed membrane fraction. In both cases, the COXII peptide is soluble under mild conditions in a nonionic detergent and is precipitable by antibodies to subunit II. The production of subunit II in the in vitro translation system is stimulated as strongly by addition of soybean phospholipid vesicles as by microsomal membranes, providing further evidence of membrane insertion and stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Structurally unique plant cytochrome c oxidase isolated from wheat germ, a rich source of plant mitochondrial enzymes.
Purification and characterization of plant cytochrome c oxidases have been impeded by the difficulty of obtaining enough plant mitochondria. We have found commercial wheat germ to be a rich and convenient source of mitochondrial membranes containing respiratory chain complexes in ratios and amounts similar to mitochondria prepared from etiolated seedlings. Cytochrome c oxidase was purified from these membranes by anion-exchange (MonoQ) fast protein liquid chromatography. The enzyme is highly active (turnover number up to 1000 s-1) and exhibits biphasic cytochrome c reaction kinetics similar to those of beef heart oxidase. As with other plant oxidases, the visible spectrum of wheat germ oxidase in the reduced form is blue-shifted compared to other eukaryotic cytochrome oxidases, with peaks at 441 and 602 nm. The electron paramagnetic resonance spectrum of CuA of the wheat germ enzyme is very similar to that of the maize and beef heart enzymes, suggesting that the copper environment is not altered. Sodium dodecyl sulfate-polyacrylamide gels show a subunit composition in which subunits I-IV resemble those of the yeast enzyme in size and antigenicity, while three to four smaller peptides are dissimilar to yeast and other eukaryotic oxidases. A difference between the subunit composition of the wheat germ and wheat seedling enzymes suggests the existence of a developmental or tissue-specific form of cytochrome oxidase in plants.
Animals↗
Hemes a and a3 environments of plant cytochrome c oxidase.
The structures of hemes a and a3 of maize and wheat germ cytochrome c oxidase were investigated by resonance Raman spectroscopy. Comparison between the plant and mammalian cytochrome oxidases revealed that (i) the vinyl groups associated with hemes a and a3 vibrate at higher frequencies in the plant enzyme than in the mammalian enzyme, suggesting different degrees of interaction between the heme cores and their periphery; (ii) aside from the geometry of the vinyl group, the structure of heme a3 in plant cytochrome oxidase is essentially unchanged from that of its mammalian counterpart; (iii) the vibrational band associated with the formyl group of reduced heme a shows relatively weak enhancement in the Soret-excited resonance Raman spectra of maize and wheat germ cytochrome oxidase, suggesting that the formyl group of ferrous heme a in the plant enzymes is conjugated only slightly to the porphyrin ring; and (iv) for oxidized heme a, the formyl vibration is strongly enhanced, but its frequency indicates a weaker interaction with the protein milieu relative to the mammalian enzyme. These observations suggest that the local environment around the formyl position of the heme a chromophore differs in the plant and mammalian cytochrome oxidases. The implication of the latter feature in the mechanism of proton pumping by cytochrome oxidase is discussed.
Electron Transport Complex IV↗