Cytochrome Bo Research Articles

The dinuclear center of cytochrome bo3 from Escherichia coli.

For the study of the dinuclear center of heme-copper oxidases cytochrome bo3 from Escherichia coli offers several advantages over the extensively characterized bovine cytochrome c oxidase. The availability of strains with enhanced levels of expression allows purification of the significant amounts of enzyme required for detailed spectroscopic studies. Cytochrome bo3 is readily prepared as the fast form, with a homogeneous dinuclear center which gives rise to characteristic broad EPR signals not seen in CcO. The absence of CuA and the incorporation of protohemes allows for a detailed interpretation of the MCD spectra arising from the dinuclear center heme o3. Careful analysis allows us to distinguish between small molecules that bind to heme o3, those which are ligands of CuB, and those which react to yield higher oxidation states of heme o3. Here we review results from our studies of the reactions of fast cytochrome bo3 with formate, fluoride, chloride, azide, cyanide, NO, and H2O2.

Matrix-assisted laser desorption ionization mass spectrometry of membrane proteins: Demonstration of a simple method to determine subunit molecular weights of hydrophobic subunits

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry has been used to obtain accurate molecular weight information for each subunit of several hydrophobic integral membrane proteins: cytochrome bo 3 (4 subunits) and cytochrome bd (2 subunits) from E. coli, and the be 1 complex (3 subunits) and the cytochrome c oxidase (3 subunits) from Rhodobacter sphaeroides. The results demonstrate that the MALDI method is a convenient, quick, sensitive and reliable means for obtaining the molecular masses of the subunits of purified multisubunit membrane proteins.

Fast cytochrome bo from Escherichia coli binds two molecules of nitric oxide at CuB.

The reaction of nitric oxide (NO) with fast cytochrome bo from Escherichia coli has been studied by electronic absorption, MCD, and EPR spectroscopy. Titration of the enzyme with NO showed the formation of two distinct species, consistent with NO binding stoichiometries of 1:1 and 2:1 with observed dissociation constants at pH 7.5 of approximately 2.3 x 10(-)6 and 3.3 x 10(-)5 M. Monitoring the titration by EPR spectroscopy revealed that the broad EPR signals at g approximately 7.3, 3.7, and 2.8 due to magnetic interaction between high-spin heme o (S = 5/2) and CuBII (S = 1/2) are lost. A high-spin heme o signal at g = 6.0 appears as the 1:1 complex is formed but is lost again on formation of the 2:1 complex, which is EPR silent. The absorption spectrum shows that heme o remains in the high-spin FeIII state throughout the titration. These results are consistent with the binding of up to two NO molecules at CuBII. This has been confirmed by studies with the Cl- adduct of fast cytochrome bo. MCD evidence shows that heme o remains ligated by histidine and water. Addition of excess NO to the Cl- adduct leads to the appearance of a high-spin FeIII heme EPR signal. Hence chloride ion binds to CuB, blocking the binding of a second NO molecule. These results suggest a mechanism for the reduction of NO to nitrous oxide by cytochrome bo and cytochrome c oxidase in which the binding of two cis NO molecules at CuB permits the formation of an N-N bond and the abstraction of oxygen by the heme group.

A conserved glutamic acid in helix VI of cytochrome bo3 influences a key step in oxygen reduction.

We have compared the reactions with dioxygen of wild-type cytochrome bo3 and a mutant in which a conserved glutamic acid at position-286 of subunit I has been changed to an alanine. Flow-flash experiments reveal that oxygen binding and the rate of heme-heme electron transfer are unaffected by the mutation. Reaction of the fully (3-electron) reduced mutant cytochrome bo3 with dioxygen yields a binuclear center which is substantially in the P (peroxy) state, not the well-characterized F (oxyferryl) state which is the product of the reaction of the fully reduced wild-type enzyme with dioxygen [Puustinen, A., et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 1545-1548]. These results confirm that proton uptake is important in controlling the later stages of dioxygen reduction in heme-copper oxidases and show that E286 is an important component of the channel that delivers these protons to the active site.

Expression of the Escherichia coli bo-Type Ubiquinol Oxidase with a Chimeric Subunit II Having the CuA-Cytochrome c Domain from the Thermophilic Bacillus caa3-Type Cytochrome c Oxidase

The C-terminal periplasmic domain of subunit II of the Escherichia coli bo-type ubiquinol oxidase was replaced with the counterpart of the thermophilic Bacillus caa3-type cytochrome c oxidase containing the CuA-cytochrome c domain by means of gene engineering techniques. The chimeric terminal oxidase was expressed by a pBR322 derivative in a terminal oxidase deficient mutant of E. coli, although the amount of the chimeric enzyme was smaller than that of the Escherichia coli bo-type ubiquinol oxidase expressed by the original cytochrome bo-expressing plasmid. The chimeric enzyme showed much higher TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) oxidase activity than the wild-type cytochrome bo, but lower activity than the thermophilic Bacillus caa3-type cytochrome c oxidase. The chimeric subunit II was confirmed to bind to heme C. These results suggest that the CuA-cytochrome c domain grafted to this membrane anchor can facilitate electron transfer from reduced TMPD to low-spin protoheme b in subunit I.

Fourier transform infrared evidence for connectivity between CuB and glutamic acid 286 in cytochrome bo3 from Escherichia coli.

Photodissociation of fully reduced, carbonmonoxy cytochrome bo3 causes ultrafast transfer of carbon monoxide (C triple bond O) from heme iron to CuB in the binuclear site. At low temperatures, the C triple bond O remains bound to CuB for extended times. Here, we show that the binding of C triple bond O to CuB perturbs the IR stretch of an un-ionized carboxylic acid residue, which is identified as Glu286 by mutation to Asp or to Cys. Before photodissociation, the carbonyl (C=O)-stretching frequency of this carboxylic acid residue is 1726 cm-1 for Glu286 and 1759 cm-1 for Glu286Asp. These frequencies are definitive evidence for un-ionized R-COOH and suggest that the carboxylic acids are hydrogen-bonded, though more extensively in Glu286. In Glu286Cys, this IR feature is lost altogether. We ascribe the frequency shifts in the C=O IR absorptions to the effects of binding photodissociated C triple bond O to CuB, which are relay ed to the 286 locus. Conversely, the 2065 cm-1 C triple bond O stretch of CuB-CO is markedly affected by both mutations. These effects are ascribed to changes in the Lewis acidity of CuB, or to displacement of a CuB histidine ligand by C triple bond O. C triple bond O binding to CuB also induces a downshift of an IR band which can be attributed to an aromatic C-H stretch, possibly of histidine imidazole, at about 3140 cm-1. The results suggest an easily polarizable, through-bond connectivity between one of the histidine CuB ligands and the carboxylic group of Glu286. A chain of bound water molecules may provide such a connection, which is of interest in the context of the proton pump mechanism of the heme-copper oxidases.

Glutamic acid 286 in subunit I of cytochrome bo3 is involved in proton translocation.

Glutamic acid 286 (E286; Escherichia coli cytochrome bo3 numbering) in subunit I of the respiratory heme-copper oxidases is highly conserved and has been suggested to be involved in proton translocation. We report a technique of enzyme reconstitution that yields essentially unidirectionally oriented cytochrome bo3 vesicles in which proton translocation can be measured. Such experiments are not feasible in the E286Q mutant due to strong inhibition of respiration, but this is not the case for the mutants E286D and E286C. The reconstituted E286D mutant enzyme readily translocates protons whereas E286C does not. Loss of proton translocation in the D135N mutant, but not in D135E or D407N, also is verified using proteoliposomes. Stopped-flow experiments show that the peroxy intermediate accumulates in the reaction of the E286Q and E286C mutant enzymes with O2. We conclude that an acidic function of the 286 locus is essential for the mechanism of proton translocation.

Subunit II of the cytochrome bo3 ubiquinol oxidase from Escherichia coli is a lipoprotein.

The purified Escherichia coli cytochrome bo3 ubiquinol oxidase contains four subunits that are each integral components of the cytoplasmic membrane. The molecular weight of each of the subunits has been determined by matrix-assisted laser desorption ionization mass spectrometry (MALDI). The observed molecular weight of subunit II (CyoA) is considerably less than the calculated value from the deduced amino acid sequence, indicating possible posttranslational processing. The similarity of a portion of the sequence near the N-terminus of CyoA with the sequences of known prokaryotic membrane-bound lipoproteins suggested that CyoA is proteolytically processed to generate an N-terminus at Cys25, and that Cys25 is covalently modified by the addition of lipids. This would be consistent with the observed molecular mass, and was confirmed by demonstrating the incorporation of radioactive palmitic acid into subunit II of the cytochrome bo3 oxidase. Site-directed mutagenesis replacing Cys25 by alanine prevents the processing, generating a precursor form of CyoA with a higher molecular mass. The C25A mutant of CyoA still assembles as an active quinol oxidase capable of supporting growth of the cells by aerobic respiration. Hence, this unusual processing of a cytoplasmic membrane protein, which is already anchored to the membrane by two transmembrane helices, is not essential for either assembly or function.

Substitutions of charged amino acid residues conserved in subunit I perturb the redox metal centers of the Escherichia coli bo-type ubiquinol oxidase.

Cytochrome bo is a four-subunit quinol oxidase in the aerobic respiratory chain of Escherichia coli and functions as a redox-coupled proton pump. Subunit I binds all the redox metal centers, low-spin heme b, high-spin heme o, and Cu(B), and serves as a reaction center of the oxidase complex. This work focuses on the functional and structural roles of 14 charged amino acid residues that are conserved in subunit I of the heme-copper terminal oxidases. Substitutions of Lys55, Tyr173, Asp188, Asp256, Arg481, and Arg482 by neutral amino acid residues did not affect the catalytic activity and spectroscopic properties of the cytoplasmic membranes. In contrast, genetic complementation tests indicated that replacements of Arg80, Asp135, Arg257, Glu286, Tyr288, Lys362, Asp407, and Glu540 resulted in nonfunctional enzymes. The R80Q mutation caused loss of a diagnostic peak for low-spin heme b in the 77 K redox difference spectrum. The K362Q, D407N, and E540Q mutations affected the CO-binding by the heme-copper binuclear center. The D135N, R257Q, E286Q, and Y288F mutations specifically eliminated the Cu(B) center from the oxidase complex, whereas the E286D mutant did not show significant perturbations on the redox metal centers even though it was still inactive. Based on these findings and recent crystallographic studies on cytochrome c oxidases, we discuss the possible roles of the conserved charged amino acid residues in subunit I of the heme-copper terminal oxidases.

Assignment and functional roles of the cyoABCDE gene products required for the Escherichia coli bo-type quinol oxidase.

Cytochrome bo from Escherichia coli belongs to the heme-copper terminal oxidase superfamily and functions as a redox-driven proton pump. In the present study, we examined the functional roles of the cyoABCDE genes, which encode cytochrome bo. We expressed the cyoABCDE genes in minicells using pTTQ18 derivatives and identified subunits II, I, III, and IV of the oxidase complex and heme O synthase as polypeptides with molecular weights of 33,500, 75,000, 20,500, 12,000, and 28,000, respectively. The expression level of heme O synthase (CyoE) was much lower than those of the oxidase subunits and seems to be controlled just tightly enough for the incorporation of heme O into the oxidase complex. To facilitate functional analysis of the gene products, we developed a single copy expression vector pHNF2, a derivative of the F-sex factor. Genetic complementation tests showed that deletions in each gene resulted in nonfunctional enzymes. Western blotting analysis indicated that the expression levels of subunits I and II were not affected by the deletions in the other cyo gene products. However, spectroscopic analyses of the mutant membranes revealed that all the deletions perturbed or eliminated the redox metal centers in subunit I. Present findings suggest that subunits II, III, and IV of the oxidase complex are required for the assembly of the metal centers in subunit I.

EPR spectroscopy of Escherichia coli cytochrome bo which lacks Cu B

The spectroscopic and ligand-binding properties of a copper-deficient cytochrome bo 3 , a member of the haem–copper superfamily of terminal oxidases, are reported and contrasted with those of the native enzyme. The enzyme lacks the copper atom (Cu B) which is normally an integral part of the catalytic site. The consequences of loss of the Cu B are the loss of antiferromagnetic coupling to the high-spin haem and an inability to form any of the integer-spin derivatives of the enzyme. Low-spin compounds of the normally high-spin haem are still formed with appropriate ligands, although these are modified.

One-step purification of histidine-tagged cytochrome bo3 from Escherichia coli and demonstration that associated quinone is not required for the structural integrity of the oxidase

The cytochrome bo 3 ubiquinol oxidase from Escherichia coli is a member of the heme-copper superfamily of proton-pumping respiratory oxidases. An improved preparative protocol was desired that would minimize the potential damage during protein isolation of labile mutants of the oxidase. Variants of the oxidase containing a histidine tag at the carboxy-terminus of either subunit I, II or III were constructed. The constructs with the histidine tag on either subunit I or II successfully allowed the enzyme to be isolated with high purity in one step using Ni 2+ affinity chromatography. The enzyme with the histidine tag on subunit II is particularly useful insofar as the enzyme isolated in this manner has little, if any, heterogeneity resulting from the presence of heme O in the low spin heme-binding site, i.e., cytochrome oo 3 is minimized. The enzyme can be prepared in virtually any quantity very rapidly and is suitable for biophysical characterization. Cytochrome bo 3 was prepared in either Triton X-100, sucrose monolaurate, or dodecyl maltoside. The enzyme isolated in the presence of either sucrose monolaurate or dodecyl maltoside contains approximately one equivalent of associated ubiquinone, whereas this is absent when Triton X-100 is used. However, the UV/vis absorbance and steady-state kinetic properties of the enzyme are virtually identical regardless of which detergent is used. These data are consistent with previous reports that cytochrome bo 3 contains an equivalent of `tightly associated' ubiquinone, but clearly demonstrate that this quinone can be removed without damaging the enzyme and is not critical to the maintenance of the native structure of the oxidase.

Oxidation of ubiquinol by cytochrome bo3 from Escherichia coli: kinetics of electron and proton transfer.

In this study we have used the so-called flow-flash technique to investigate electron and proton transfer during the reaction between cytochrome bo3 with bound ubiquinol (QH2) and dioxygen. The results are compared to those from the well-characterized mitochondrial cytochrome alpha alpha3. Qualitatively, the same type of absorbance changes associated with electron transfer were observed in both enzymes whereas the protonation reactions were markedly different. In the bacterial QH2-bound enzyme, three kinetic phases with time constants of approximately 45 micros, approximately 700 micros, and approximately 4 ms associated with electron-transfer reactions were observed. The first phase is attributed to oxidation of hemes b and o3 and formation of the "peroxy" intermediate. The second and third phases were not observed after addition of the herbicide HQNO, which displaces QH2 from its binding site. They are attributed to electron transfer from QH2 to heme b and from heme b to the binuclear center, respectively. In both enzymes, the initial electron transfer was followed by a slower uptake of 0.9 +/- 0.3 proton per enzyme molecule (tau approximately 90 micros), previously attributed to protonation of a group near the binuclear center. Only in the bacterial enzyme, the second electron-transfer reaction was accompanied by a net release of 1.1 +/- 0.3 H+, which is attributed to proton release during oxidation of QH2. It was followed by a slower uptake of 1.2 +/- 0.4 H+ during transfer of the fourth electron to the binuclear center. The two slowest protonation reactions were not observed in the presence of HQNO.

Projection structure of the cytochrome bo ubiquinol oxidase from Escherichia coli at 6 A resolution.

The haem-copper cytochrome oxidases are terminal catalysts of the respiratory chains in aerobic organisms. These integral membrane protein complexes catalyse the reduction of molecular oxygen to water and utilize the free energy of this reaction to generate a transmembrane proton gradient. Quinol oxidase complexes such as the Escherichia coli cytochrome bo belong to this superfamily. To elucidate the similarities as well as differences between ubiquinol and cytochrome c oxidases, we have analysed two-dimensional crystals of cytochrome bo by cryo-electron microscopy. The crystals diffract beyond 5 A. A projection map was calculated to a resolution of 6 A. All four subunits can be identified and single alpha-helices are resolved within the density for the protein complex. The comparison with the three-dimensional structure of cytochrome c oxidase shows the clear structural similarity within the common functional core surrounding the metal-binding sites in subunit I. It also indicates subtle differences which are due to the distinct subunit composition. This study can be extended to a three-dimensional structure analysis of the quinol oxidase complex by electron image processing of tilted crystals.

Haem O and a putative cytochrome bo in a mutant of Bacillus cereus impaired in the synthesis of haem A.

In a spontaneous mutant (PYM1) of Bacillus cereus impaired in the synthesis of haem A, no haem-A-containing cytochromes were detected spectroscopically. The haem A deficiency was compensated by high levels of haem O and a CO-reactive cytochrome o in membranes; no other oxidases were detected. In contrast, the wild-type strain had considerable amounts of haem A and negligible levels of haem O. The mutant PYM1 exhibited normal colony morphology, growth, and sporulation in nonfermentable media, whereas on fermentable media, the mutant overproduced acid, which led to poor growth and inhibition of sporulation. External control of the pH of the medium in fermentable media allowed close-to-normal growth and massive sporulation of the mutant. The presence of membrane-bound cytochrome caa3-OII and aa3-II subunits in strain PYM1 was confirmed by Western blots and haem C staining (COII subunit). Western blotting also revealed that in contrast to the wild-type - strain PYM1 contained the membrane-bound subunits caa3-COI and aa3-I, but in low amounts. The effect of several respiratory inhibitors on the respiratory system of strain PYM1 suggested that the terminal oxidase is highly resistant to KCN and CO and that a c-type cytochrome might be involved in the electron transfer sequence to the putative cytochrome bo.

Uncompetitive substrate inhibition and noncompetitive inhibition by 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) and 2-n-nonyl-4-hydroxyquinoline-N-oxide (NQNO) is observed for the cytochrome bo3 complex: implications for a Q(H2)-loop proton translocation mechanism.

The cytochrome bo3 ubiquinol oxidase complex from Escherichia coli contains two binding sites for ubiquinone(ol) (UQ(H2)). One of these binding sites, the ubiquinol oxidation site, is clearly in dynamic equilibrium with the UQ(H2) pool in the membrane. The second site has a high affinity for ubiquinone (UQ), stabilizes a semiquinone species, and is located physically close to the low-spin heme b component of the enzyme. The UQ molecule in this site has been proposed to remain strongly bound to the enzyme during enzyme turnover and to act as a cofactor facilitating the transfer of electrons from the substrate ubiquinol to heme b [Sato-Watanabe et al. (1994) J. Biol. Chem. 269, 28908-28912]. In this paper, the steady-state turnover of the enzyme is examined in the presence and absence of inhibitors (UHDBT and NQNO) that appear to be recognized as ubisemiquinone analogs. It is found that the kinetics are accounted for best by a noncompetitive inhibitor binding model. Furthermore, at high concentrations, the substrates ubiquinol-1 and ubiquinol-2 inhibit turnover in an uncompetitive fashion. Together, these observations strongly suggest that there must be at least two UQ(H2) binding sites that are in rapid equilibrium with the UQ(H2) pool under turnover conditions. Although these data do not rule out the possibility that a strongly bound UQ molecule functions to facilitate electron transfer to heme b, they are more consistent with the behavior expected if the two UQ(H2) binding sites were to function in a Q(H2)-loop mechanism (similar to that of the cytochrome bc1 complex) as originally proposed by Musser and co-workers [(1993) FEBS Lett. 327, 131-136]. In this model, ubiquinol is oxidized at one site and ubiquinone is reduced at the second site. While the structural similarities of the heme-copper ubiquinol and cytochrome c oxidase complexes suggest the possibility that these two families of enzymes translocate protons by similar mechanisms, the current observations indicate that the Q(H2)-loop proton translocation mechanism for the heme-copper ubiquinol oxidase complexes should be further investigated and experimentally tested.

Q-band Electron Nuclear Double Resonance (ENDOR) and X-band EPR of the Sulfobetaine 12 Heat-treated Cytochrome c Oxidase Complex

Heat treatment of the bovine cytochrome c oxidase complex in the zwitterionic detergent sulfobetaine 12 (SB-12) results in loss of subunit III and the appearance of a type II copper center as characterized by electron paramagnetic resonance (EPR) spectroscopy. Previous authors (Nilsson, T., Copeland, R. A., Smith, P. A., and Chan, S. I. (1988) Biochemistry 27, 8254-8260) have interpreted this type II copper center as a modified version of the CuA site. By using electron nuclear double resonance spectroscopy, it is found that the CuA proton and nitrogen resonances remain present in the SB-12 heat-treated enzyme and that three new nitrogen resonances appear having hyperfine coupling constants consistent with histidine ligation. These hyperfine coupling constants correlate well with those recently found for the CuB histidines from the cytochrome aa(3)-600 quinol oxidase from Bacillus subtilis (Fann, Y. C., Ahmed, I., Blackburn, N. J., Boswell, J. S., Verkhovskaya, M. L., Hoffman, B. M., and Wikstr9om, M. (1995) Biochemistry 34, 10245-10255). In addition, the total EPR-detectable copper concentration per enzyme molecule approximately doubles upon SB-12 heat treatment. Finally, the observed type II copper EPR spectrum is virtually indistinguishable from the EPR spectrum of CuB of the as-isolated cytochrome bo3 complex from Escherichia coli. These data indicate that the type II copper species that appears results from a breaking of the strong antiferromagnetic coupling of the heme a3-CuB binuclear center.

The reaction of Escherichia coli cytochrome bo with H 20 2: Evidence for the formation of an oxyferryl species by two distinct routes

We have re-examined the reaction of fast oxidised cytochrome bo with H 2O 2 in a stopped-flow spectrophotometer. Monitoring the reaction at 582 nm allows us to observe the formation and decay of a spectroscopically distinct intermediate which accumulates transiently prior to the formation of an oxyferryl species previously characterised in this laboratory (Watmough, N.J., Cheesman, M.R., Greenwood, C. and Thomson, A.J. (1994) Biochem. J. 300, 469–475 [1]). The reaction shows three distinct phases of which the fast and intermediate phases are bimolecular and show a marked pH dependence. Initially these results appeared incompatible with the report that only one equivalent of H 2O 2 is required to generate the oxyferryl species (Moody, A.J. and Rich, P.R. (1994) Eur. J. Biochem. 226, 731–737 [2]). However, these data can be reconciled by a branched reaction mechanism whose contributions differ according to the peroxide concentration used.

Redox FTIR difference spectroscopy using caged electrons reveals contributions of carboxyl groups to the catalytic mechanism of haemcopper oxidases

Redox spectra of the haem-copper oxidases cytochrome aa 3 of Rhodobacter sphaeroides and cytochrome bo 3 of Escherichia coli were recorded in the visible and infrared spectral regions. The reduction of oxidases was initiated after light activation of the ‘caged electron’ donor riboflavin. Infrared redox difference spectra exhibit absorbance changes in the amide I region, which are indicative of very small redox-finked conformational movements in the polypeptide backbone. A reproducible redox-dependent pattern of positive and negative absorption changes is found in the carbonyl region (1680–1750 cm −1). The carbonyl bands shift to lower frequencies due to isotope exchange of the solvent H 2O to D 2O. This common feature of cytochrome c and quinol oxidases indicates that at least (i) one redox-sensitive carboxyl group is in the protonated state in the oxidized form and (ii) one carboxylic acid is involved at a catalytic step — presumably in proton translocation — of haem-copper oxidases.

Purification and characterization of the succinate dehydrogenase complex and CO-reactive b-type cytochromes from the facultative alkaliphile Bacillus firmus OF4

The presence of a bo-type terminal oxidase in Bacillus firmus OF4 had been suggested from the effects of CO on the spectra of reduced membrane cytochromes (Hicks, D.B., Plass, R.J. and Quirk, P.G. (1991) J. Bacteriol. 173, 5010–5016). In that study the CO-binding b-type cytochrome was partially purified by anion exchange chromatography. No further purification was attempted but later HPLC analysis indicated the absence of significant heme O in the B. firmus OF4 membranes. The current work shows that the partially purified cytochrome b is actually composed of three different b-type cytochromes which can be separated and purified by a combination of ion-exchange, hydroxyapatite and gel filtration chromatographies. Two of the cytochromes were CO-reactive but lacked the characteristic multisubunit composition of known terminal oxidases. Neither purified cytochrome catalyzed quinol or ferrocytochrome c oxidation. The more abundant CO-reactive b-type cytochrome (cytochrome b 560) had an apparent molecular mass of 10 kDa, whereas the other, more minor component (cytochrome b 558), was partially purified and showed two bands of 23 and 17 kDa on SDS-PAGE. The functions of the cytochromes b 560 and b 558 remain unknown but together they account for the spectrum originally attributed to cytochrome bo. The third, non-CO reactive, cytochrome b, was associated with substantial succinate dehydrogenase activity and was purified as a three subunit succinate dehydrogenase complex with high specific activity (17.7 μmol/min/mg). Limited N-terminal sequence of each subunit demonstrated marked similarity to the complex from Bacillus subtilis. The cytochrome b of the alkaliphile enzyme was reduced about 50% by succinate compared to the level of reduction achieved by dithionite. The enzyme reacted with both napthoquinones and benzoquinones. The results presented indicate that Bacillus firmus OF4 contains a succinate dehydrogenase complex with very similar properties to the enzyme from Bacillus subtilis, but does not contain a cytochrome o-type terminal oxidase under the growth conditions studied.