Ca(2+)-binding site in Rhodobacter sphaeroides cytochrome C oxidase

Cytochrome c oxidase (COX) from R. sphaeroides contains one Ca(2+) ion per enzyme that is not removed by dialysis versus EGTA. This is similar to COX from Paracoccus denitrificans [Pfitzner, U., Kirichenko, A., Konstantinov, A. A., Mertens, M., Wittershagen, A., Kolbesen, B. O., Steffens, G. C. M., Harrenga, A., Michel, H., and Ludwig, B. (1999) FEBS Lett. 456, 365-369] and is in contrast to the bovine oxidase, which binds Ca(2+) reversibly. A series of R. sphaeroides mutants with replacements of the E54, Q61, and D485 residues, which form the Ca(2+) coordination sphere in subunit I, has been generated. The substitutions for the E54 residue do not assemble normally. Mutants with the Q61 replacements are active and retain the tightly bound Ca(2+); their spectra are not perturbed by added Ca(2+) or EGTA. The D485A mutant is active, binds to Ca(2+) reversibly, like the mitochondrial oxidase, and exhibits the red shift in the heme a absorption spectrum upon Ca(2+) binding for both reduced and oxidized states of heme a. The K(d) value of 6 nM determined by equilibrium titrations is much lower than that reported for the homologous D477A mutant of Paracoccus denitrificans or for bovine COX (K(d) = 1-3 microM). The rate of Ca(2+) binding with the D485A oxidase (k(on) = 5 x 10(3) M(-1) s(-1)) is comparable to that observed earlier for bovine COX, but the off-rate is extremely slow (approximately 10(-3) s(-1)) and highly temperature-dependent. The k(off) /k(on) ratio (190 nM) is about 30-fold higher than the equilibrium K(d) of 6 nM, indicating that formation of the Ca(2+)-adduct may involve more than one step. Sodium ions reverse the Ca(2+)-induced red shift of heme a and dramatically decrease the rate of Ca(2+) binding to the D485A mutant COX. With the D485A mutant, 1 Ca(2+) competes with 1 Na(+) for the binding site, whereas 2 Na(+) compete with 1 Ca(2+) for binding to the bovine oxidase. This finding indicates that the aspartic residue D442 (a homologue of R. sphaeroides D485) may be the second Na(+) binding site in bovine COX. No effect of Ca(2+) binding to the D485A mutant is evident on either the steady-state enzymatic activity or several time-resolved partial steps of the catalytic cycle. It is proposed that the tightly bound Ca(2+) plays a structural role in the bacterial oxidases while the reversible binding with the mammalian enzyme may be involved in the regulation of mitochondrial function.     

Substitutions for glutamate 101 in subunit II of cytochrome c oxidase from Rhodobacter sphaeroides result in blocking the proton-conducting K-channel

Two functional input pathways for protons have been characterized in the heme-copper oxidases: the D-channel and the K-channel. These two proton-conducting channels have different functional roles and have been defined both by X-ray crystallography and by the characterization of site-directed mutants. Whereas the entrance of the D-channel is well-defined as D132(I) (subunit I; Rhodobacter sphaeroides numbering), the entrance of the K-channel has not been clearly defined. Previous mutagenesis studies of the cytochrome bo(3) quinol oxidase from Escherichia coli implicated an almost fully conserved glutamic acid residue within subunit II as a likely candidate for the entrance of the K-channel. The current work examines the properties of mutants of this conserved glutamate in the oxidase from R. sphaeroides (E101(II)I,A,C,Q,D,N,H) and residues in the immediate vicinity of E101(II). It is shown that virtually any substitution for E101(II), including E101(II)D, strongly reduces oxidase turnover (to 8-29%). Furthermore, the low steady-state activity correlates with an inhibition of the rate of reduction of heme a(3) prior to the reaction with O(2). These are phenotypes expected of K-channel mutants. It is concluded that the predominant entry point for protons going into the K-channel of cytochrome oxidase is the surface-exposed glutamic acid E101(II) in subunit II.     

A mutation in subunit I of cytochrome oxidase from Rhodobacter sphaeroides results in an increase in steady-state activity but completely eliminates proton pumping

The heme-copper oxidases convert the free energy liberated in the reduction of O(2) to water into a transmembrane proton electrochemical potential (protonmotive force). One of the essential structural elements of the enzyme is the D-channel, which is thought to be the input pathway, both for protons which go to form H(2)O ("chemical protons") and for protons that get translocated across the lipid membrane ("pumped protons"). The D-channel contains a chain of water molecules extending about 25 A from an aspartic acid (D132 in the Rhodobacter sphaeroides oxidase) near the cytoplasmic ("inside") enzyme surface to a glutamic acid (E286) in the protein interior. Mutations in which either of these acidic residues is replaced by their corresponding amides (D132N or E286Q) result in severe inhibition of enzyme activity. In the current work, an asparagine located in the D-channel has been replaced by the corresponding acid (N139 to D; N98 in bovine enzyme) with dramatic consequences. The N139D mutation not only completely eliminates proton pumping but, at the same time, confers a substantial increase (150-300%) in the steady-state cytochrome oxidase activity. The N139D mutant of the R. sphaeroides oxidase was further characterized by examining the rates of individual steps in the catalytic cycle. Under anaerobic conditions, the rate of reduction of heme a(3) in the fully oxidized enzyme, prior to the reaction with O(2), is identical to that of the wild-type oxidase and is not accelerated. However, the rate of reaction of the fully reduced enzyme with O(2) is accelerated by the N139D mutation, as shown by a more rapid F --> O transition. Whereas the rates of formation and decay of the oxygenated intermediates are altered, the nature of the oxygenated intermediates is not perturbed by the N139D mutation.     

Probing the access of protons to the K pathway in the Paracoccus denitrificans cytochrome c oxidase

In recent studies on heme-copper oxidases a particular glutamate residue in subunit II has been suggested to constitute the entry point of the so-called K pathway. In contrast, mutations of this residue (E78(II)) in the Paracoccus denitrificans cytochrome c oxidase do not affect its catalytic activity at all (E78(II)Q) or reduce it to about 50% (E78(II)A); in the latter case, the mutation causes no drastic decrease in heme a(3) reduction kinetics under anaerobic conditions, when compared to typical K pathway mutants. Moreover, both mutant enzymes retain full proton-pumping competence. While oxidized-minus-reduced Fourier-transform infrared difference spectroscopy demonstrates that E78(II) is indeed addressed by the redox state of the enzyme, absence of variations in the spectral range characteristic for protonated aspartic and glutamic acids at approximately 1760 to 1710 cm(-1) excludes the protonation of E78(II) in the course of the redox reaction in the studied pH range, although shifts of vibrational modes at 1570 and 1400 cm(-1) reflect the reorganization of its deprotonated side chain at pH values greater than 4.8. We therefore conclude that protons do not enter the K channel via E78(II) in the Paracoccus enzyme.     

Proton pumping mechanism of bovine heart cytochrome c oxidase

X-ray structures of bovine heart cytochrome c oxidase at 1.8/1.9 A resolution in the oxidized/reduced states exhibit a redox coupled conformational change of an aspartate located near the intermembrane surface of the enzyme. The alteration of the microenvironment of the carboxyl group of this aspartate residue indicates the occurrence of deprotonation upon reduction of the enzyme. The residue is connected with the matrix surface of the enzyme by a hydrogen-bond network that includes heme a via its propionate and formyl groups. These X-ray structures provide evidence that proton pumping occurs through the hydrogen bond network and is driven by the low spin heme. The function of the aspartate is confirmed by mutation of the aspartate to asparagine. Although the amino acid residues of the hydrogen bond network and the structures of the low spin heme peripheral groups are not completely conserved amongst members of the heme-copper terminal oxidase superfamily, the existence of low spin heme and the hydrogen bond network suggests that the low spin heme provides the driving element of the proton-pumping process.     

Electron and proton transfer in the arginine-54-methionine mutant of cytochrome c oxidase from Paracoccus denitrificans

Arginine 54 in subunit I of cytochrome c oxidase from Paracoccus denitrificans interacts with the formyl group of heme a. Mutation of this arginine to methionine (R54M) dramatically changes the spectral properties of heme a and lowers its midpoint redox potential [Kannt et al. (1999) J. Biol. Chem. 274, 37974-37981; Lee et al. (2000) Biochemistry 39, 2989-2996; Riistama et al. (2000) Biochim. Biophys. Acta 1456, 1-4]. During anaerobic reduction of the mutant enzyme, a small fraction of heme a is reduced first along with heme a(3), while most of heme a is reduced later. This suggests that electron transfer is impaired thermodynamically due to the low redox potential of heme a but that it still takes place from Cu(A) via heme a to the binuclear site as in wild-type enzyme, with no detectable bypass from Cu(A) directly to the binuclear site. Consistent with this, the proton translocation efficiency is unaffected at 1 H(+)/e(-) in the mutant enzyme, although turnover is strongly inhibited. Time-resolved electrometry shows that when the fully reduced enzyme reacts with O(2), the fast phase of membrane potential generation during the P(R )()--> F transition is unaffected by the mutation, whereas the slow phase (F --> O transition) is strongly decelerated. In the 3e(-)-reduced mutant enzyme heme a remains oxidized due to its lowered midpoint potential, whereas Cu(A) and the binuclear site are reduced. In this case the reaction with O(2) proceeds via the P(M) state because transfer of the electron from Cu(A) to the binuclear site is delayed. The single phase of membrane potential generation in the 3e(-)-reduced mutant enzyme, which thus corresponds to the P(M)--> F transition, is decelerated, but its amplitude is comparable to that of the P(R)--> F transition. From this we conclude that the completely (4e(-)) reduced enzyme is fully capable of proton translocation.     

Cytochrome c oxidase as a calcium binding protein. Studies on the role of a conserved aspartate in helices XI-XII cytoplasmic loop in cation binding

The aa(3)-type cytochrome c oxidases from mitochondria and bacteria contain a cation-binding site located in subunit I near heme a. In the oxidases from Paracoccus denitrificans or Rhodobacter sphaeroides, the site is occupied by tightly bound calcium, whereas the mitochondrial oxidase binds reversibly calcium or sodium that compete with each other. The functional role of the site has not yet been established. D477A mutation in subunit I of P. denitrificans oxidase converts the cation-binding site to a mitochondrial-type form that binds reversibly calcium and sodium ions [Pfitzner, U., Kirichenko, A., et al. (1999) FEBS Lett. 456, 365-369]. We have studied reversible cation binding with P. denitrificans D477A oxidase and compared it with that in bovine enzyme. In bovine oxidase, one Ca(2+) competes with two Na(+) for the binding, indicating the presence of two Na(+)-binding sites in the enzyme, Na(+)((1)) and Na(+)((2)). In contrast, the D477A mutant of COX from P. denitrificans reveals competition of Ca(2+) (K(d) = 1 microM) with only one sodium ion (K(d) = 4 mM). The second binding site for Na(+) in bovine oxidase is proposed to involve D442, homologous to D477 in P. denitrificans oxidase. A putative place for Na(+)((2)) in subunit I of bovine oxidase has been found with the aid of structure modeling located 7.4 A from the bound Na(+)((1)) . Na(+)((2)) interacts with a cluster of residues forming an exit part of the so-called H-proton channel, including D51 and S441.     

Cloning, sequencing and deletion from the chromosome of the gene encoding subunit I of the aa3-type cytochrome c oxidase of Rhodobacter sphaeroides

The ctaD gene encoding subunit I of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides has been cloned. The gene encodes a polypeptide of 565 residues which is highly homologous to the sequences of subunit I from other prokaryotic and eukaryotic sources, e.g. 51% identity with that from bovine, and 75% identity with that from Paracoccus denitrificans. The ctaD gene was deleted from the chromosome of R. sphaeroides, resulting in a strain that spectroscopically lacks cytochrome a. This strain maintains about 50% of the cytochrome c oxidase activity of the wild-type strain owing to the presence of an alternate o-type cytochrome c oxidase. The aa3-type oxidase was restored by complementing the chromosomal deletion with a plasmid-borne copy of the ctaD gene. This system is well suited for site-directed mutagenesis probing of the structure and function of cytochrome c oxidase.     

Glutamate 107 in subunit I of the cytochrome bd quinol oxidase from Escherichia coli is protonated and near the heme d/heme b595 binuclear center

Cytochrome bd is a quinol oxidase from Escherichia coli, which is optimally expressed under microaerophilic growth conditions. The enzyme catalyzes the two-electron oxidation of either ubiquinol or menaquinol in the membrane and scavenges O2 at low concentrations, reducing it to water. Previous work has shown that, although cytochrome bd does not pump protons, turnover is coupled to the generation of a proton motive force. The generation of a proton electrochemical gradient results from the release of protons from the oxidation of quinol to the periplasm and the uptake of protons used to form H2O from the cytoplasm. Because the active site has been shown to be located near the periplasmic side of the membrane, a proton channel must facilitate the delivery of protons from the cytoplasm to the site of water formation. Two conserved glutamic acid residues, E107 and E99, are located in transmembrane helix III in subunit I and have been proposed to form part of this putative proton channel. In the current work, it is shown that mutations in either of these residues results in the loss of quinol oxidase activity and can result in the loss of the two hemes at the active site, hemes d and b595. One mutant, E107Q, while being totally inactive, retains the hemes. Fourier transform infrared (FTIR) redox difference spectroscopy has identified absorption bands from the COOH group of E107. The data show that E107 is protonated at pH 7.6 and that it is perturbed by the reduction of the heme d/heme b595 binuclear center at the active site. In contrast, mutation of an acidic residue known to be at or near the quinol-binding site (E257A) also inactivates the enzyme but has no substantial influence on the FTIR redox difference spectrum. Mutagenesis shows that there are several acidic residues, including E99 and E107 as well as D29 (in CydB), which are important for the assembly or stability of the heme d/heme b595 active site.     

Glutamate-89 in subunit II of cytochrome bo3 from Escherichia coli is required for the function of the heme-copper oxidase

Recent electrostatics calculations on the cytochrome c oxidase from Paracoccus denitrificans revealed an unexpected coupling between the redox state of the heme-copper center and the state of protonation of a glutamic acid (E78II) that is 25 A away in subunit II of the oxidase. Examination of more than 300 sequences of the homologous subunit in other heme-copper oxidases shows that this residue is virtually totally conserved and is in a cluster of very highly conserved residues at the "negative" end (bacterial cytoplasm or mitochondrial matrix) of the second transmembrane helix. The functional importance of several residues in this cluster (E89II, W93II, T94II, and P96II) was examined by site-directed mutagenesis of the corresponding region of the cytochrome bo(3) quinol oxidase from Escherichia coli (where E89II is the equivalent of residue E78II of the P. denitrificans oxidase). Substitution of E89II with either alanine or glutamine resulted in reducing the rate of turnover to about 43 or 10% of the wild-type value, respectively, whereas E89D has only about 60% of the activity of the control oxidase. The quinol oxidase activity of the W93V mutant is also reduced to about 30% of that of the wild-type oxidase. Spectroscopic studies with the purified E89A and E89Q mutants indicate no perturbation of the heme-copper center. The data suggest that E89II (E. coli numbering) is critical for the function of the heme copper oxidases. The proximity to K362 suggests that this glutamic acid residue may regulate proton entry or transit through the K-channel. This hypothesis is supported by the finding that the degree of oxidation of the low-spin heme b is greater in the steady state using hydrogen peroxide as an oxidant in place of dioxygen for the E89Q mutant. Thus, it appears that the inhibition resulting from the E89II mutation is due to a block in the reduction of the heme-copper binuclear center, expected for K-channel mutants.     

Arginine 391 in subunit I of the cytochrome bd quinol oxidase from Escherichia coli stabilizes the reduced form of the hemes and is essential for quinol oxidase activity

The cytochrome bd quinol oxidase is one of two respiratory oxidases in Escherichia coli. It oxidizes dihydroubiquinol or dihydromenaquinol while reducing dioxygen to water. The bd-type oxidases have only been found in prokaryotes and have been implicated in the survival of some bacteria, including pathogens, under conditions of low aeration. With a high affinity for dioxygen, cytochrome bd not only couples respiration to the generation of a proton motive force but also scavenges O(2). In the current work, the role of a highly conserved arginine residue is explored by site-directed mutagenesis. Four mutations were made: R391A, R391K, R391M, and R391Q. All of the mutations except R391K result in enzyme lacking ubiquinol oxidase activity. Oxidase activity using the artificial reductant N,N,N',N'-tetramethyl-p-phenylenediamine in place of ubiquinol was, however, unimpaired by the mutations, indicating that the catalytic center where O(2) is reduced is intact. UV-visible spectra of each of the mutant oxidases show no perturbations to any of the three heme components (heme b(558), heme b(595), and heme d). However, spectroelectrochemical titrations of the R391A mutant reveal that the midpoint potentials of all of the heme components are substantially lower compared with the wild type enzyme. Since Arg(391) is close to Met(393), one of the axial ligands to heme b(558), it is to be expected that the R391A mutation might destabilize the reduced form of heme b(558). The fact that the midpoint potentials of heme d and heme b(595) are also significantly lowered in the R391A mutant is consistent with these hemes being physically close together on the periplasmic side of the membrane.     

The role of the cross-link His-Tyr in the functional properties of the binuclear center in cytochrome c oxidase

Resonance Raman and Fourier transform infrared spectroscopies have been used to study the aa(3)-type cytochrome c oxidase and the Y280H mutant from Paracoccus denitrificans. The stability of the binuclear center in the absence of the Tyr(280)-His(276) cross-link is not compromised since heme a(3) retains the same proximal environment, spin, and coordination state as in the wild type enzyme in both the oxidized and reduced states. We observe two C-O modes in the Y280H mutant at 1966 and 1975 cm(-1). The 1975 cm(-1) mode is assigned to a gamma-form and represents a structure of the active site in which Cu(B) exerts a steric effect on the heme a(3)-bound CO. Therefore, the role of the cross-link is to fix Cu(B) in a certain configuration and distance from heme a(3), and not to allow histidine ligands to coordinate to Cu(B) rather than to heme a(3), rendering the enzyme inactive, as proposed recently (Das, T. K., Pecoraro, C., Tomson, F. L., Gennis, R. B., and Rousseau, D. L. (1998) Biochemistry 37, 14471-14476). The results provide solid evidence that in the Y280H mutant the catalytic site retains its active configuration that allows O(2) binding to heme a(3). Oxygenated intermediates are formed by mixing oxygen with the CO-bound mixed-valence wild type and Y280H enzymes with similar Soret maxima at 438 nm.     

Characterization of heme-binding properties of Paracoccus denitrificans Surf1 proteins

Biogenesis of cytochrome c oxidase (COX) is a highly complex process involving >30 chaperones in eukaryotes; those required for the incorporation of the copper and heme cofactors are also conserved in bacteria. Surf1, associated with heme a insertion and with Leigh syndrome if defective in humans, is present as two homologs in the soil bacterium Paracoccus denitrificans, Surf1c and Surf1q. In an in vitro interaction assay, the heme a transfer from purified heme a synthase, CtaA, to Surf1c was followed, and both Surf proteins were tested for their heme a binding properties. Mutation of four strictly conserved amino acid residues within the transmembrane part of each Surf1 protein confirmed their requirement for heme binding. Interestingly the mutation of a tryptophan residue in transmembrane helix II (W200 in Surf1c and W209 in Surf1q) led to a drastic switch in the heme composition, with Surf1 now being populated mostly by heme o, the intermediate in the heme a biosynthetic pathway. This tryptophan residue discriminates between the two heme moieties, apparently coordinates the formyl group of heme a, and most likely presents the cofactor in a spatial orientation suitable for optimal transfer to its target site within subunit I of cytochrome c oxidase.     

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.     

Nitric oxide reacts with the single-electron reduced active site of cytochrome c oxidase

The reduction kinetics of the mutants K354M and D124N of the Paracoccus denitrificans cytochrome oxidase (heme aa(3)) by ruthenium hexamine was investigated by stopped-flow spectrophotometry in the absence/presence of NO. Quick heme a reduction precedes the biphasic heme a(3) reduction, which is extremely slow in the K354M mutant (k(1) = 0.09 +/- 0.01 s(-1); k(2) = 0.005 +/- 0.001 s(-1)) but much faster in the D124N aa(3) (k(1) = 21 +/- 6 s(-1); k(2) = 2.2 +/- 0.5 s(-1)). NO causes a very large increase (>100-fold) in the rate constant of heme a(3) reduction in the K354M mutant but only a approximately 5-fold increase in the D124N mutant. The K354M enzyme reacts rapidly with O(2) when fully reduced but is essentially inactive in turnover; thus, it was proposed that impaired reduction of the active site is the cause of activity loss. Since at saturating [NO], heme a(3) reduction is approximately 100-fold faster than the extremely low turnover rate, we conclude that, contrary to O(2), NO can react not only with the two-electron but also with the single-electron reduced active site. This mechanism would account for the efficient inhibition of cytochrome oxidase activity by NO in the wild-type enzyme, both from P. denitrificans and from beef heart. Results also suggest that the H(+)-conducting K pathway, but not the D pathway, controls the kinetics of the single-electron reduction of the active site.     

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.     

G204D, a mutation that blocks the proton-conducting D-channel of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides

Cytochrome c oxidase uses the free energy of oxygen reduction to establish a transmembrane proton gradient. The proton-conducting D-channel in this enzyme is the major input pathway for protons which go to the binuclear center for water formation ("chemical protons") and likely the only input pathway for protons that get translocated across the lipid membrane ("pumped protons"). The D-channel starts at an acidic residue near the protein surface (D132, Rhodobacter sphaeroides numbering) and leads to another acidic residue near the binuclear center. Recent studies have shown that mutants that introduce an additional acidic residue in the channel (N139D) have the remarkable effect of accelerating steady-state oxidase activity but completely eliminating proton pumping. In this work, an aspartic acid was introduced at the position of glycine 204, G204D, which is also within the D-channel, and the effects were examined. In contrast to N139D, the G204D mutation results in a dramatic decrease of the steady-state oxygen reductase activity (<2% of wild type) [Aagaard, A., and Brzezinski, P. (2001) FEBS Lett. 494, 157-160]. The residual activity is not coupled to the proton pump, and furthermore, in reconstituted vesicles the mutant enzyme exhibits a reverse respiration control ratio; i.e., the mutant oxidase activity is stimulated rather than inhibited when working against a protonmotive force. Hence, the mutant behaves very much like the D132N, which blocks proton uptake through the D-channel. Single-turnover experiments show that the rate-limiting step in the reaction of O2 with the fully reduced G204D mutant is the F --> O transition, similar to the D132N mutant. The block of the D-channel in the D132N mutant can be partly bypassed by biochemically removing subunit III from the enzyme, indicating that removal of the subunit reveals an alternate entrance for protons to the channel. However, this is not observed with the G204D mutant. This suggests that the cryptic entrance to the D-channel that is revealed by the removal of subunit III is between the levels of G204 and D132.     

Site-directed mutation of the highly conserved region near the Q-loop of the cytochrome bd quinol oxidase from Escherichia coli specifically perturbs heme b595.

Cytochrome bd is one of the two quinol oxidases in the respiratory chain of Escherichia coli. The enzyme contains three heme prosthetic groups. The dioxygen binding site is heme d, which is thought to be part of the heme-heme binuclear center along with heme b(595), which is a high-spin heme whose function is not known. Protein sequence alignments [Osborne, J. P., and Gennis, R. B. (1999) Biochim. Biophys Acta 1410, 32--50] of cytochrome bd quinol oxidase sequences from different microorganisms have revealed a highly conserved sequence (GWXXXEXGRQPW; bold letters indicate strictly conserved residues) predicted to be on the periplasmic side of the membrane between transmembrane helices 8 and 9 in subunit I. The functional importance of this region is investigated in the current work by site-directed mutagenesis. Several mutations in this region (W441A, E445A/Q, R448A, Q449A, and W451A) resulted in a catalytically inactive enzyme with abnormal UV--vis spectra. E445A was selected for detailed analysis because of the absence of the absorption bands from heme b(595). Detailed spectroscopic and chemical analyses, indeed, show that one of the three heme prosthetic groups in the enzyme, heme b(595), is specifically perturbed and mostly missing from this mutant. Surprisingly, heme d, while known to interact with heme b(595), appears relatively unperturbed, whereas the low-spin heme b(558) shows some modification. This is the first report of a mutation that specifically affects the binding site of heme b(595).     

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.