The purification and characterization of the cytochrome d terminal oxidase complex of the Escherichia coli aerobic respiratory chain

The aerobic respiratory chain of Escherichia coli is branched. In aerobically grown cells harvested in midexponential phase, a respiratory chain containing only b-type cytochromes is predominant. This chain contains a terminal oxidase which is a b-type cytochrome, referred to as cytochrome o. However, when the bacteria are grown under conditions of oxygen limitation, additional components of the respiratory chain are induced, as evidenced by the appearance of new spectroscopic species. These include a new b-type cytochrome, cytochrome b558, as well as cytochrome a1 and cytochrome d. In this paper, a purification protocol and the initial characterization of the terminal oxidase complex containing cytochrome d are reported. Solubilization of the membrane is effected by Zwittergent 3-12, and purification is accomplished by chromatography with DEAE-Sepharose CL-6B and hydroxyapatite. The complex contains cytochrome b558, a1, and d. Analysis by sodium dodecyl sulfate-polyacrylamide gels indicates that the complex contains only two types of polypeptides with the molecular weights estimated to be 57,000 and 43,000. The purified complex has oxidase activity in the presence of detergents, utilizing substrates including ubinquinol-1, N,N,N',N'-tetramethyl-p-phenylenediamine, and 2,3,5,6-tetramethyl-p-phenylenediamine. The cytochrome d complex contains protoheme IX and iron, but does not contain nonheme iron or copper. Approximately half of the cytochromes which are thought to participate in E. coli aerobic respiration are accounted for by this single complex. These results suggest that the E. coli aerobic respiratory chain is organized around a relatively small number of cytochrome-containing complexes.     

Two hemes in Bacillus subtilis succinate:menaquinone oxidoreductase (complex II).

Succinate:menaquinone-7 oxidoreductase (complex II) of the Gram-positive bacterium Bacillus subtilis consists of equimolar amounts of three polypeptides; a 65-kDa FAD-containing polypeptide, a 28-kDa iron-sulfur cluster containing polypeptide, and a 23-kDa membrane-spanning cytochrome b558 polypeptide. The enzyme complex was overproduced 2-3-fold in membranes of B. subtilis cells containing the sdhCAB operon on a low copy number plasmid and was purified in the presence of detergent. The cytochrome b558 subunit alone was similarly overexpressed in a complex II deficient mutant and partially purified. Isolated complex II catalyzed the reduction of various quinones and also quinol oxidation. Both activities were efficiently albeit not completely blocked by 2-n-heptyl-4-hydroxyquinoline N-oxide. Chemical analysis demonstrated two protoheme IX per complex II. One heme component was found to have an Em,7.4 of +65 mV and an EPR gmax signal at 3.68, to be fully reducible by succinate, and showed a symmetrical alpha-band absorption peak at 555 nm at 77 K. The other heme component was found to have an Em,7.4 of -95 mV and an EPR gmax signal at 3.42, was not reducible by succinate under steady-state conditions, and showed in the reduced state an apparent split alpha-band absorption peak with maxima at 553 and 558 nm at 77 K. Potentiometric titrations of partially purified cytochrome b558 subunit demonstrated that the isolated cytochrome b558 also contains two hemes. Some of the properties, i.e., the alpha-band light absorption peak at 77 K, the line shapes of the EPR gmax signals, and reactivity with carbon monoxide were observed to be different in B. subtilis cytochrome b558 isolated and in complex II. This suggests that the bound flavoprotein and iron-sulfur protein subunits protect or affect the heme environment in the assembled complex.     

Location of heme axial ligands in the cytochrome d terminal oxidase complex of Escherichia coli determined by site-directed mutagenesis

The cytochrome d terminal oxidase complex is one of two terminal oxidases which are components of the aerobic respiratory chain of Escherichia coli. This membrane-bound enzyme catalyzes the two-electron oxidation of ubiquinol and the four-electron reduction of oxygen to water. Enzyme turnover generates proton and voltage gradients across the bilayer. The oxidase is a heterodimer containing 2 mol of protoheme IX and 1 or 2 mol of heme d per mol of complex. To explain the functional properties of the enzyme, a simple model has been proposed in which it is speculated that the heme prosthetic groups define two separate active sites on opposite sides of the membrane at which the oxidation of quinol and the reduction of water, respectively, are catalyzed. This paper represents an initial effort to define the axial ligands of each of the three or four hemes within the amino acid sequence of the oxidase subunits. Each of the 10 histidine residues has been altered by site-directed mutagenesis with the expectation that histidine residues are likely candidates for heme ligands. Eight of the 10 histidine residues are not essential for enzyme activity, and 2 appear to function as heme axial ligands. Histidine 186 in subunit I is required for the cytochrome b558 component of the enzyme. This residue is likely to be located near the periplasmic surface of the membrane. Histidine 19, near the amino terminus of subunit I also appears to be a heme ligand. It is concluded that two of the four or five expected heme axial ligands have been tentatively identified, although further work is required to confirm these conclusions. A minimum of two additional axial ligands must be residues other than histidine.     

Purification and characterization of the cytochrome bd complex from Azotobacter vinelandii: comparison to the complex from Escherichia coli.

Partial purification of a cytochrome bd complex from Azotobacter vinelandii grown under high aeration was achieved by isolating respiratory particles enriched in this hemoprotein via differential centrifugation and detergent extraction. The cytochrome bd complex was subsequently solubilized from the inner membrane with dodecyl maltoside and purified to near homogeneity via DEAE-Sepharose chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the complex consisted of two subunits, with sizes in good agreement with those predicted from the cloned cyd locus (59.7 and 42 kDa). Spectral analysis of the purified complex indicated that the heme components present were cytochromes b560, b595, and d; CO difference spectral studies identified cytochrome d as a CO-reactive component. The complex had a Km for ubiquinol-1 approximately seven times larger than that for the analogous bd complex from Escherichia coli, and O2 consumption curves revealed a Km value for O2 three times greater than that which we determined for the E. coli bd complex.     

Specific overproduction and purification of the cytochrome b558 component of the cytochrome d complex from Escherichia coli

In Escherichia coli strain GR84N[pNG10], the cloned gene for subunit I of the membrane-bound cytochrome d complex resulted in the overproduction of cytochrome b558 and facilitated purification of this cytochrome. Extracting membranes with 1% Triton X-100 followed by two chromatographic steps yielded a single band on sodium dodecyl sulfate-polyacrylamide gels corresponding to subunit I (Mr 57 000). Purified cytochrome b558 was in its native state as determined by difference absorption spectroscopy and by potentiometric analysis. Both the membranes of strain GR84N[pNG10] and the purified subunit I lacked the other two spectroscopically defined cytochromes, b595 (previously "a1") and d, of the cytochrome d complex. Reconstitution of cytochrome b558 in phospholipid vesicles demonstrated that cytochrome b558 can be reduced by ubiquinol but that it does not reduce molecular oxygen. Heme extraction of cytochrome b558 yielded an extinction coefficient of 22 000 M-1 cm-1 for the wavelength pair of 560 and 580 nm in the reduced-minus-oxidized spectrum. The mutation on pNG10 that eliminates subunit II was mapped to a 250 base pair DNA fragment.     

Oxoferryl-porphyrin radical catalytic intermediate in cytochrome bd oxidases protects cells from formation of reactive oxygen species

The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b(558) that donates electrons to a binuclear heme b(595)/heme d center. The reaction with O(2) and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O(2), the O-O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b(595). Compound I accumulates to 0.75-0.85 per enzyme in agreement with its much higher rate of formation (~20,000 s(-1)) compared with its rate of decay (~1,900 s(-1)). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b(558) before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O-O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O-O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species.     

Heme O biosynthesis in Escherichia coli: the cyoE gene in the cytochrome bo operon encodes a protoheme IX farnesyltransferase.

The cytochrome bo complex of Escherichia coli is encoded by the cyoABCDE operon and functions as a redox-coupled proton pump. In this study, we have constructed eight cyoE deletion mutants and found that all the mutants were nonfunctional. Spectroscopic and heme analyses of the mutant oxidases revealed that the mutations specifically substituted protoheme IX for heme O present in the high-spin heme binding site. We found also that the overexpression of the cyoE gene in a cyo operon deletion strain resulted in a conversion of protoheme IX to heme O. Since the CyoE protein contains the putative allylic polyprenyldiphosphate binding domain, we concluded that the cyoE gene encodes a novel enzyme, protoheme IX farnesyltransferase, essential for heme O biosynthesis.     

Interaction of cytochrome bd with carbon monoxide at low and room temperatures: evidence that only a small fraction of heme b595 reacts with CO

Azotobacter vinelandii is an obligately aerobic bacterium in which aerotolerant dinitrogen fixation requires cytochrome bd. This oxidase comprises two polypeptide subunits and three hemes, but no copper, and has been studied extensively. However, there remain apparently conflicting reports on the reactivity of the high spin heme b(595) with ligands. Using purified cytochrome bd, we show that absorption changes induced by CO photodissociation from the fully reduced cytochrome bd at low temperatures demonstrate binding of the ligand with heme b(595). However, the magnitude of these changes corresponds to the reaction with CO of only about 5% of the heme. CO binding with a minor fraction of heme b(595) is also revealed at room temperature by time-resolved studies of CO recombination. The data resolve the apparent discrepancies between conclusions drawn from room and low temperature spectroscopic studies of the CO reaction with cytochrome bd. The results are consistent with the proposal that hemes b(595) and d form a diheme oxygen-reducing center with a binding capacity for a single exogenous ligand molecule that partitions between the hemes d and b(595) in accordance with their intrinsic affinities for the ligand. In this model, the affinity of heme b(595) for CO is about 20-fold lower than that of heme d.     

Assignment of the histidine axial ligands to the cytochrome bH and cytochrome bL components of the bc1 complex from Rhodobacter sphaeroides by site-directed mutagenesis.

The cytochrome b subunit of the bc1 complex contains two cytochrome components, cytochrome bH and cytochrome bL. Sequence comparisons of this polypeptide from a number of organisms have revealed four invariant histidines which have been postulated to be the heme ligands for the two protoheme IX prosthetic groups. In Rhodobacter sphaeroides, these correspond to His97, His111, His198, and His212. In this paper, the results of amino acid substitutions at each of these positions are reported. Replacement of His97 by either Asp or Asn and of His198 by Asn or Tyr resulted in loss of both cytochrome components. However, His111Asn, His111Asp, and His212Asp all resulted in the selective loss of cytochrome bH and the retention of cytochrome bL. Furthermore, flash kinetics studies show that the myxothiazol-sensitive quinol oxidase (Qz) site associated with cytochrome bL is still functional. These data support the assignment of the axial ligands to cytochrome bH (His111 and His212) and cytochrome bL (His97 and His198). This pairing is consistent with current models of the cytochrome b subunit with eight transmembrane alpha-helices.     

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).     

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.     

Superoxide production by cytochrome b558 purified from neutrophils in a reconstituted system with an exogenous reductase.

Cytochrome b558, which is considered to be an essential component of the phagocytic superoxide (O2-)-generating system, was highly purified from porcine neutrophils. The isolated cytochrome was resolved into two polypeptides with molecular masses of 60-90 and 19 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For enzymatic reduction of purified cytochrome b558, we utilized hepatic NADPH-cytochrome P450 reductase purified from rat liver microsomes. More than 80% of the cytochrome was reduced by incubation with the reductase and NADPH under the anaerobic condition, and was quickly reoxidized by the air. As indicated by measurement of oxygen consumption, the purified cytochrome catalytically reduced oxygen at a rate equal to approximately 30% of the activity of the phorbol myristate acetate-activated cells on the basis of cytochrome b558 content. Electron paramagnetic resonance study with a spin trapping agent 5, 5-dimethyl-1-pyrroline-1-oxide demonstrated that O2- is the exclusive primary product in the reduction of oxygen by the cytochrome. This gives direct evidence that cytochrome b558 functions as the terminal oxidizing enzyme in the O2- -generating system of neutrophils. This also establishes a new functional class of heme proteins that catalyzes one-electron reduction of molecular oxygen.     

Purification and some properties of the small subunit of cytochrome b558 from human neutrophils

We have attempted to purify the heme moiety of cytochrome b558 from human neutrophils. Cytochrome b558 was solubilized from the crude membrane fraction which was pretreated with both 1 M potassium phosphate buffer and 1% octyl glucoside at low ionic strength. The solubilization of cytochrome b558 was carried out efficiently with 1.6% octyl glucoside in the presence of 100 mM phosphate buffer. Solubilized cytochrome b558 was purified by hydroxylapatite, DEAE-Sephacel, and Mono Q fast protein liquid chromatography. The specific content of purified cytochrome b558 was 37 nmol/mg of protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified cytochrome b558 revealed a single band of 20,000 Da. The large subunit of cytochrome b558, which has been reported by others, could not be found in purified cytochrome b558 even with silver staining. The amino acid composition of the heme-containing moiety of cytochrome b558 was abundant in hydrophobic amino acids. The circular dichroism spectra of both oxidized and reduced b558-type cytochromes exhibited bilobed bands with wavelengths of crossover points closely corresponding to those of the maxima in the optical absorbance spectra at the Soret region. Furthermore, there were some differences in the shoulders and peak widths of CD spectra between oxidized and reduced b558-type cytochromes. These results indicate that this method provides the purification of the small subunit of human cytochrome b558 which is the heme-carrying subunit of cytochrome b558, and suggest that cytochrome b558 has heme-heme interaction and some conformational changes in the alternation of the redox state.     

Characterization of the cytochrome d terminal oxidase complex of Escherichia coli using polyclonal and monoclonal antibodies

The cytochrome d terminal oxidase complex is one of two terminal oxidases in the aerobic respiratory chain of Escherichia coli. Previous work has shown by dodecyl sulfate-polyacrylamide gel electrophoresis that this enzyme contains two subunits (I and II) and three cytochrome components, b558 , a1, and d. Reconstitution studies have demonstrated that the enzyme functions as a ubiquinol-8 oxidase and catalyzes an electrogenic reaction, i.e. turnover is accompanied by a charge separation across the membrane bilayer. In this paper, monoclonal and polyclonal antibodies were used to obtain structural information about the cytochrome d complex. It is shown that antibodies directed against subunit I effectively inhibit ubiquinol-1 oxidation by the purified enzyme in detergent, whereas antibodies which bind to subunit II have no effect on quinol oxidation. The oxidation rate of N,N,N',N'-tetramethyl-p-phenylenediamine, in contrast, is unaffected by antisubunit I antibodies, but is inhibited by antibodies against subunit II. It is concluded that the quinol oxidation site is on subunit I, previously shown to be the cytochrome b558 component of the complex, and that N,N,N',N'-tetramethyl-p-phenylenediamine oxidation occurs at a secondary site on subunit II. The antibodies were also used to analyze the results of a protein cross-linking experiment. Dimethyl suberimidate was used to cross-link the subunits of purified, solubilized oxidase. Immunoblot analysis of the products of this cross-linking clearly indicate that subunit II probably exists as a dimer within the complex. Finally, it is shown that the purified enzyme contains tightly bound lipopolysaccharide. This was revealed after discovering that one of the monoclonal antibodies raised against the purified complex is actually directed against lipopolysaccharide. The significance of this finding is not known.     

Identification of subunit I as the cytochrome b558 component of the cytochrome d terminal oxidase complex of Escherichia coli

The cytochrome d terminal oxidase complex was recently purified from Escherichia coli membranes (Miller, M. J., and Gennis , R. B. (1983) J. Biol. Chem. 258, 9159-1965). The complex contains two polypeptides, subunits I and II, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and three spectroscopically defined cytochromes, b558 , a1, and d. A mutant that failed to oxidize N,N,N',N'-tetramethyl-p-phenylenediamine was obtained which was lacking this terminal oxidase complex and was shown to map at a locus called cyd on the E. coli genome. In this paper, localized mutagenesis was used to generate a series of mutants in the cytochrome d terminal oxidase. These mutants were isolated by a newly developed selection procedure based on their sensitivity to azide. Two classes of mutants which map to the cyd locus were obtained, cydA and cydB . The cydA phenotype included the lack of all three spectroscopically detectable cytochromes as well as the absence of both polypeptides, determined by immunological criteria. Strains manifesting the cydB phenotype lacked cytochromes a1 and d, but had a normal amount of cytochrome b558 . Immunological analysis showed that subunit I (57,000 daltons) was present in the membranes, but that subunit II (43,000 daltons) was missing. These data justify the conclusion that subunit I of this two-subunit complex can be identified as the cytochrome b558 component of the cytochrome d terminal oxidase complex.     

Reconstitution of superoxide-forming NADPH oxidase activity with cytochrome b558 purified from porcine neutrophils. Requirement of a membrane-bound flavin enzyme for reconstitution of activity.

Cytochrome b558 of pig blood neutrophils was purified from the membranes of resting cells to examine its ability to reconstitute superoxide (O2-)-forming NADPH oxidase activity in a cell-free assay system containing cytosol and fatty acid. The membrane-associated cytochrome b558 was solubilized with a detergent, n-heptyl beta-thioglucoside, and purified by DEAE-Sepharose, heparin-Sepharose, and Mono Q column chromatography. The final preparation of cytochrome containing 11.5 nmol of protoheme/mg of protein gave bands of the large and small subunits on immunoblotted gel. The cell-free system with the purified cytochrome alone as a membrane component showed little O2(-)-generating activity in the absence of exogenous FAD. However, the system showed high O2(-)-generating activity of 31.8 mol/s/mol of cytochrome b558 (52.5% of the original O2(-)-generating activity of the solubilized membranes) in the presence of a nitro blue tetrazolium (NBT) reductase fraction that was separated from the cytochrome b fraction by heparin-Sepharose chromatography. Heat treatment of the NBT reductase fraction resulted in loss of the O2(-)-generating activity in the reconstituted system. The O2(-)-forming activity of the reconstituted system was markedly decreased by removal of FAD from the NBT reductase fraction and was restored by readdition of FAD to the FAD-depleted reductase. The reconstituted system containing purified cytochrome b558 plus the NBT reductase showed approximately 100 times higher O2(-)-generating activity than a system containing rabbit liver NADPH-cytochrome P-450 reductase instead. These results suggest that both the FAD-dependent NBT reductase and cytochrome b558 are required as membrane redox components for O2(-)-forming NADPH oxidase activity. The present data are discussed in comparison with previously reported results on reconstituted systems containing added free FAD.     

Electron transfer process in cytochrome bd-type ubiquinol oxidase from Escherichia coli revealed by pulse radiolysis

Cytochrome bd is a two-subunit ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli and binds hemes b558, b595, and d as the redox metal centers. Taking advantage of spectroscopic properties of three hemes which exhibit distinct absorption peaks, we investigated electron transfer within the enzyme by the technique of pulse radiolysis. Reduction of the hemes in the air-oxidized, resting-state enzyme, where heme d exists in mainly an oxygenated form and partially an oxoferryl and a ferric low-spin forms, occurred in two phases. In the faster phase, radiolytically generated N-methylnicotinamide radicals simultaneously reduced the ferric hemes b558 and b595 with a second-order rate constant of 3 x 10(8) M-1 s-1, suggesting that a rapid equilibrium occurs for electron transfer between two b-type hemes long before 10 micros. In the slower phase, an intramolecular electron transfer from heme b to the oxoferryl and the ferric heme d occurred with the first-order rate constant of 4.2-5.6 x 10(2) s-1. In contrast, the oxygenated heme d did not exhibit significant spectral change. Reactions with the fully oxidized and hydrogen peroxide-treated forms demonstrated that the oxidation and/or ligation states of heme d do not affect the heme b reduction. The following intramolecular electron transfer transformed the ferric and oxoferryl forms of heme d to the ferrous and ferric forms, respectively, with the first-order rate constants of 3.4 x 10(3) and 5.9 x 10(2) s-1, respectively.     

Heme/heme redox interaction and resolution of individual optical absorption spectra of the hemes in cytochrome bd from Escherichia coli

Cytochrome bd is a terminal component of the respiratory chain of Escherichia coli catalyzing reduction of molecular oxygen to water. It contains three hemes, b₅₅₈, b₅₉₅, and d. The detailed spectroelectrochemical redox titration and numerical modeling of the data reveal significant redox interaction between the low-spin heme b₅₅₈ and high-spin heme b₅₉₅, whereas the interaction between heme d and either hemes b appears to be rather weak. However, the presence of heme d itself decreases much larger interaction between the two hemes b. Fitting the titration data with a model where redox interaction between the hemes is explicitly included makes it possible to extract individual absorption spectra of all hemes. The α- and β-band reduced-minus-oxidized difference spectra agree with the data published earlier ([22] J.G. Koland, M.J. Miller, R.B. Gennis, Potentiometric analysis of the purified cytochrome d terminal oxidase complex from Escherichia coli, Biochemistry 23 (1984) 1051-1056., and [23] R.M. Lorence, J.G. Koland, R.B. Gennis, Coulometric and spectroscopic analysis of the purified cytochrome d complex of Escherichia coli: evidence for the identification of “cytochrome a₁” as cytochrome b₅₉₅, Biochemistry 25 (1986) 2314-2321.). The Soret band spectra show λ_max = 429.5 nm, λ_min ≈ 413 nm (heme b₅₅₈), λ_max = 439 nm, λ_min ≈ 400 ± 1 nm (heme b₅₉₅), and λ_max = 430 nm, λ_min = 405 nm (heme d). The spectral contribution of heme d to the complex Soret band is much smaller than those of either hemes b; the Soret/α (ΔA₄₃₀:ΔA₆₂₉) ratio for heme d is 1.6.     

Catalytic intermediates of cytochrome bd terminal oxidase at steady-state: ferryl and oxy-ferrous species dominate

The cytochrome bd ubiquinol oxidase from Escherichia coli couples the exergonic two-electron oxidation of ubiquinol and four-electron reduction of O(2) to 2H(2)O to proton motive force generation by transmembrane charge separation. The oxidase contains two b-type hemes (b(558) and b(595)) and one heme d, where O(2) is captured and converted to water through sequential formation of a few intermediates. The spectral features of the isolated cytochrome bd at steady-state have been examined by stopped-flow multiwavelength absorption spectroscopy. Under turnover conditions, sustained by O(2) and dithiothreitol (DTT)-reduced ubiquinone, the ferryl and oxy-ferrous species are the mostly populated catalytic intermediates, with a residual minor fraction of the enzyme containing ferric heme d and possibly one electron on heme b(558). These findings are unprecedented and differ from those obtained with mammalian cytochrome c oxidase, in which the oxygen intermediates were not found to be populated at detectable levels under similar conditions [M.G. Mason, P. Nicholls, C.E. Cooper, The steady-state mechanism of cytochrome c oxidase: redox interactions between metal centres, Biochem. J. 422 (2009) 237-246]. The data on cytochrome bd are consistent with the observation that the purified enzyme has the heme d mainly in stable oxy-ferrous and ferryl states. The results are here discussed in the light of previously proposed models of the catalytic cycle of cytochrome bd.     

Binding of FAD to cytochrome b558 is facilitated during activation of the phagocyte NADPH oxidase, leading to superoxide production

The superoxide-producing phagocyte NADPH oxidase can be reconstituted in a cell-free system. The activity of NADPH oxidase is dependent on FAD, but the physiological status of FAD in the oxidase is not fully elucidated. To clarify the role of FAD in NADPH oxidase, FAD-free full-length recombinant p47(phox), p67(phox), p40(phox), and Rac were prepared, and the activity was reconstituted with these proteins and purified cytochrome b(558) (cyt b(558)) with different amounts of FAD. A remarkably high activity, over 100 micromol/s/micromol heme, was obtained in the oxidase with purified cyt b(558), ternary complex (p47-p67-p40(phox)), and Rac. From titration with FAD of the activity of NADPH oxidase reconstituted with purified FAD-devoid cyt b, the dissociation constant K(d) of FAD in cyt b(558) of reconstituted oxidase was estimated as nearly 1 nm. We also examined addition of FAD on the assembly process in reconstituted oxidase. The activity was remarkably enhanced when FAD was present during assembly process, and the efficacy of incorporating FAD into the vacant FAD site in purified cyt b(558) increased, compared when FAD was added after assembly processes. The absorption spectra of reconstituted oxidase under anaerobiosis showed that incorporation of FAD into cyt b(558) recovered electron flow from NADPH to heme. From both K(d) values of FAD and the amount of incorporated FAD in cyt b(558) of reconstituted oxidase, in combination with spectra, we propose the model in which the K(d) values of FAD in cyt b(558) is changeable after activation and FAD binding works as a switch to regulate electron transfer in NADPH oxidase.