Title: Cyanide-binding Site of bd-type Ubiquinol Oxidase from Escherichia coli
Abstract: We extended our investigation on the structure of the redox centers of bd-type ubiquinol oxidase from Escherichia coli using cyanide as a monitoring probe. We found that addition of cyanide to the air-oxidized O2-bound enzyme caused appearance of an infrared C-N stretching band at 2161 cm- and concomitant disappearance of the 647 nm absorption band of the cytochrome d (Fe2+)-O2 species. Addition of cyanide to the air-oxidized CO-bound enzyme also resulted in disappearance of the 635 nm absorption band and the 1983.4 cm- C-O infrared band of the cytochrome d (Fe2+)-CO species. The resulting species had a derivative-shaped electron paramagnetic resonance signal at g = 3.15. Upon partial reduction with sodium dithionite, this species was converted partly to a transient heme d (Fe3+)-C=N species having an electron paramagnetic resonance signal at gz = 2.96 and a C-N infrared band at 2138 cm-. These observations suggest that the active site of the enzyme has a heme-heme binuclear metal center distinct from that of the heme-copper terminal oxidase and that the treatment of the air-oxidized enzyme with cyanide resulted in a cyanide-bridging species with "heme d(Fe3+)-C=N-heme b595(Fe3+)" structure. We extended our investigation on the structure of the redox centers of bd-type ubiquinol oxidase from Escherichia coli using cyanide as a monitoring probe. We found that addition of cyanide to the air-oxidized O2-bound enzyme caused appearance of an infrared C-N stretching band at 2161 cm- and concomitant disappearance of the 647 nm absorption band of the cytochrome d (Fe2+)-O2 species. Addition of cyanide to the air-oxidized CO-bound enzyme also resulted in disappearance of the 635 nm absorption band and the 1983.4 cm- C-O infrared band of the cytochrome d (Fe2+)-CO species. The resulting species had a derivative-shaped electron paramagnetic resonance signal at g = 3.15. Upon partial reduction with sodium dithionite, this species was converted partly to a transient heme d (Fe3+)-C=N species having an electron paramagnetic resonance signal at gz = 2.96 and a C-N infrared band at 2138 cm-. These observations suggest that the active site of the enzyme has a heme-heme binuclear metal center distinct from that of the heme-copper terminal oxidase and that the treatment of the air-oxidized enzyme with cyanide resulted in a cyanide-bridging species with "heme d(Fe3+)-C=N-heme b595(Fe3+)" structure. INTRODUCTIONEscherichia coli has two terminal ubiquinol oxidases in the aerobic electron transfer chain: bo-type ubiquinol oxidase, which is expressed under high oxygen tension, and bd-type ubiquinol oxidase, which predominates under low oxygen tension(1Anraku Y. Annu. Rev. Biochem. 1988; 57: 101-132Crossref PubMed Scopus (170) Google Scholar, 2Poole R.K. Biochim. Biophys. Acta. 1983; 726: 205-243Crossref PubMed Scopus (206) Google Scholar). These oxidases are structurally unrelated, but both catalyze the two-electron oxidation of ubiquinol-8 and the four-electron reduction of dioxygen to produce water(1Anraku Y. Annu. Rev. Biochem. 1988; 57: 101-132Crossref PubMed Scopus (170) Google Scholar, 2Poole R.K. Biochim. Biophys. Acta. 1983; 726: 205-243Crossref PubMed Scopus (206) Google Scholar). The E. coli bd-type ubiquinol oxidase has been isolated and was found to consist of two polypeptide chains: subunit I (58 kDa) and subunit II (43 kDa)(3Kita K. Konishi K. Anraku Y. J. Biol. Chem. 1984; 259: 3375-3381Abstract Full Text PDF PubMed Google Scholar, 4Miller M.J. Gennis R.B. J. Biol. Chem. 1983; 258: 9159-9165Abstract Full Text PDF PubMed Google Scholar). The cydAB genes coding for both polypeptides have been cloned and sequenced(5Green G.N. Fang H. Lin R.-J. Newton G. Mather M. Georgiou C.D. Gennis R.B. J. Biol. Chem. 1988; 263: 13138-13143Abstract Full Text PDF PubMed Google Scholar). Within this enzyme, it has been claimed, there are three types of cytochrome heme species based on the optical spectra: cytochrome d, cytochrome b595, and cytochrome b558(6Koland J.G. Miller M.J. Gennis R.B. Biochemistry. 1984; 23: 1051-1056Crossref Scopus (48) Google Scholar, 7Lorence R.M. Koland J.G. Gennis R.B. Biochemistry. 1986; 25: 2314-2321Crossref PubMed Scopus (82) Google Scholar). Cytochrome d has a chlorin chromophore (heme D) (8Timkovich R. Cork M.S. Gennis R.B. Johnson P.Y. J. Am. Chem. Soc. 1985; 107: 6069-6075Crossref Scopus (102) Google Scholar) exhibiting a characteristic absorption maximum at 628 nm in the fully reduced state and is a primary binding site for exogenous ligands(3Kita K. Konishi K. Anraku Y. J. Biol. Chem. 1984; 259: 3375-3381Abstract Full Text PDF PubMed Google Scholar, 4Miller M.J. Gennis R.B. J. Biol. Chem. 1983; 258: 9159-9165Abstract Full Text PDF PubMed Google Scholar, 9Hata A. Kirino Y. Matsuura K. Itoh S. Hiyama T. Konishi K. Kita K. Anraku Y. Biochim. Biophys. Acta. 1985; 810: 62-72Crossref PubMed Scopus (44) Google Scholar). The dioxygen molecule forms a very stable adduct with ferrous cytochrome d showing its Fe2+-O2 stretching vibration at 568 cm- (essentially identical to that of myoglobin)(10Kahlow M.A. Loehr T.M. Zuberi T.M. Gennis R.B. J. Am. Chem. Soc. 1993; 115: 5845-5846Crossref Scopus (45) Google Scholar), and thus the enzyme in the air-oxidized state is actually an oxygenated form. The cytochrome d moiety also forms a remarkably stable oxoferryl (Fe4+=O) adduct(11Kahlow M.A. Zuberi T.M. Gennis R.B. Loehr T.M. Biochemistry. 1991; 30: 11485-11489Crossref PubMed Scopus (64) Google Scholar). Subunit I contains cytochrome b558 that shows its α and β peaks at 562 and 532 nm, respectively, in the reduced state (12Green G.N. Kranz R.G. Lorence R.M. Gennis R.B. J. Biol. Chem. 1984; 259: 7994-7997Abstract Full Text PDF PubMed Google Scholar) and most likely forms the ubiquinol-8 binding site(13Yang F.-D. Yu L. Yu C.-A. Lorence R.M. Gennis R.B. J. Biol. Chem. 1986; 261: 14987-14990Abstract Full Text PDF PubMed Google Scholar). Cytochrome b595 is an unusual b-type cytochrome. It was suggested that the optical spectrum of this cytochrome is very similar to that of high spin cytochrome c peroxidase, and its α and β bands are at 595 and 562 nm, respectively, in the reduced minus oxidized difference spectrum(7Lorence R.M. Koland J.G. Gennis R.B. Biochemistry. 1986; 25: 2314-2321Crossref PubMed Scopus (82) Google Scholar). Recent low temperature CO photolysis experiments monitored by the infrared C-O stretching absorption suggest that cytochrome d and cytochrome b595 form a binuclear center(14Hill J.J. Alben J.O. Gennis R.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5863-5867Crossref PubMed Scopus (108) Google Scholar, 15Hill J.J. Gennis R.B. Alben J.O. Biophys. J. 1994; 66 (abstr.): 367Google Scholar).In a previous study we analyzed the purified bd-type ubiquinol oxidase by resonance Raman, FT-IR, ( 1The abbreviations used are: FT-IRFourier-transform infraredEPRelectron paramagnetic resonance.) and EPR spectroscopies (16Tsubaki M. Uno T. Hori H. Mogi T. Nishimura Y. Anraku Y. FEBS Lett. 1993; 335: 13-17Crossref PubMed Scopus (15) Google Scholar). We found that heme d-bound C-O and Fe2+-CO stretching frequencies were at 1980.7 and 471 cm-, respectively, in the fully reduced state. These values differ considerably from those of the heme-copper respiratory oxidases and the oxygen-carrying hemoproteins, both groups possessing a His residue as the heme axial ligand(17Tsubaki M. Srivastava R.B. Yu N.-T. Biochemistry. 1982; 21: 1132-1140Crossref PubMed Scopus (181) Google Scholar, 18Argade P.V. Ching Y. Rousseau D.L. Science. 1984; 225: 329-331Crossref PubMed Scopus (65) Google Scholar, 19Uno T. Nishimura Y. Tsuboi M. Kita K. Anraku Y. J. Biol. Chem. 1985; 260: 6755-6760Abstract Full Text PDF PubMed Google Scholar, 20Uno T. Mogi T. Tsubaki M. Nishimura Y. Anraku Y. J. Biol. Chem. 1994; 269: 11912-11920Abstract Full Text PDF PubMed Google Scholar). EPR analyses on the air-oxidized O2-bound and Na2S2O4-reduced nitric oxide-bound forms revealed that there is no superhyperfine structure originating from the heme axial 14N ligand in the central resonance of the nitric oxide EPR signals(16Tsubaki M. Uno T. Hori H. Mogi T. Nishimura Y. Anraku Y. FEBS Lett. 1993; 335: 13-17Crossref PubMed Scopus (15) Google Scholar). These results suggest strongly that the heme d axial ligand of bd-type ubiquinol oxidase is either a His residue in an anomalous condition or some other residue making the molecular structure around the oxygen-binding site different from those of the heme-copper respiratory oxidases. Indeed, electron nuclear double resonance spectroscopy has shown that the axial ligand to heme d is most probably not a histidine or other strong nitrogenous ligand(21Jiang F.S. Zuberi T.M. Cornelius J.B. Clarkson R.B. Gennis R.B. Belford R.L. J. Am. Chem. Soc. 1993; 115: 10293-10299Crossref Scopus (1) Google Scholar). In the present study we extended our investigation on the redox centers of the E. coli bd-type ubiquinol oxidase using cyanide as a monitoring probe.EXPERIMENTAL PROCEDURESPurification of bd-type Ubiquinol OxidaseThe E. coli bd-type ubiquinol oxidase was isolated from the cytoplasmic membranes as described (22Tsubaki M. Mogi T. Anraku Y. Hori H. Biochemistry. 1993; 32: 6065-6072Crossref PubMed Scopus (81) Google Scholar) with the slight modification that Tris-HCl buffer was replaced by sodium phosphate buffer and that magnesium chloride was omitted during solubilization. The strain GR84N/pNG2 (cyo+cydA2 recA/cydAB+ Tetr(23Green G.N. Kranz J.E. Gennis R.B. Gene (Amst.). 1984; 32: 99-106Crossref PubMed Scopus (33) Google Scholar)), a kind gift from R. B. Gennis, which can overproduce bd-type ubiquinol oxidase, was used. The purified enzyme in 50 mM sodium phosphate (pH 7.4) containing 0.1% sucrose monolaurate SM-1200 (Mitsubishi-Kasei Food Corp., Tokyo) was stored at -80°C until use.Measurements of EPR, FT-IR, and Optical SpectraEPR measurements were carried out at X-band (9.23 GHz) microwave frequency with a home-built EPR spectrometer as described previously (16Tsubaki M. Uno T. Hori H. Mogi T. Nishimura Y. Anraku Y. FEBS Lett. 1993; 335: 13-17Crossref PubMed Scopus (15) Google Scholar, 22Tsubaki M. Mogi T. Anraku Y. Hori H. Biochemistry. 1993; 32: 6065-6072Crossref PubMed Scopus (81) Google Scholar) and a Varian E-12 EPR spectrometer equipped with an Oxford flow cryostat (ESR-900). Infrared spectra were recorded at 4°C with a Perkin-Elmer (model 1850) FT-IR spectrophotometer as described previously (24Tsubaki M. Shinzawa K. Yoshikawa S. Biophys. J. 1992; 63: 1564-1571Abstract Full Text PDF PubMed Scopus (10) Google Scholar) or a JASCO (model VALOR-III) FT-IR spectrophotometer. Absolute optical spectra of bd-type ubiquinol oxidase in the infrared cells with a 51-μm path length were measured at room temperature with a UVIKON 860 UV-visible spectrophotometer (Kontron Instruments, Inc.) before and after FT-IR measurements.MiscellaneousProtein concentration was determined using the BCA protein assay reagent (Pierce). Metal contents were determined by inductively coupled plasma atomic emission spectrophotometry with a SPS 1200VR plasma spectrometer (Seiko Instruments Inc., Tokyo). Heme B contents were analyzed as described previously(25Berry E.A. Trumpower B.L. Anal. Biochem. 1987; 161: 1-15Crossref PubMed Scopus (737) Google Scholar). The following potassium cyanide isotopes were used: K12C14N (natural abundance, Nacalai Tesque); K12C15N (99.4 atom % 15N, Isotec Inc.); K13C14N (99 atom % 13C, Isotec Inc.); and K13C15N (99 atom % 13C, 99 atom % 15N, Icon). Other chemicals were commercial products of analytical grade.RESULTSHeme B, Metal Contents, and Optical Extinction CoefficientsThe heme B and metal contents of the purified bd-type ubiquinol oxidase were examined. Heme B content was 18.2 ± 1.6 nmol/mg protein, whereas the heme B:Fe:Cu:Zn ratio (in mol basis) was 2:3.60 ± 0.60:0.084 ± 0.003:0.098 ± 0.026 (averages of three independent preparations). Theoretical value of the heme B content is 19.8 nmol/mg protein assuming the presence of 2 mol of heme B/mol of bd-type ubiquinol oxidase with a molecular mass of 101 kDa(3Kita K. Konishi K. Anraku Y. J. Biol. Chem. 1984; 259: 3375-3381Abstract Full Text PDF PubMed Google Scholar, 4Miller M.J. Gennis R.B. J. Biol. Chem. 1983; 258: 9159-9165Abstract Full Text PDF PubMed Google Scholar, 5Green G.N. Fang H. Lin R.-J. Newton G. Mather M. Georgiou C.D. Gennis R.B. J. Biol. Chem. 1988; 263: 13138-13143Abstract Full Text PDF PubMed Google Scholar). It was reported that bd-type ubiquinol oxidase contains only 1 mol of cytochrome d/mol of the enzyme(14Hill J.J. Alben J.O. Gennis R.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5863-5867Crossref PubMed Scopus (108) Google Scholar). Thus, previous and present heme B and metal content analyses of bd-type ubiquinol oxidase indicate that there is 1 mol of heme D and 2 mol of heme B and adventitious non-heme iron/mol of the enzyme and no other constitutive metals (including magnesium, calcium, chromium, manganese, cobalt, nickel, zinc, selenium, molybdenum, and tungsten)(4Miller M.J. Gennis R.B. J. Biol. Chem. 1983; 258: 9159-9165Abstract Full Text PDF PubMed Google Scholar). On the basis of the present analyses, the optical extinction coefficients were calculated (Table 1). The results show considerable deviations from those previously reported(3Kita K. Konishi K. Anraku Y. J. Biol. Chem. 1984; 259: 3375-3381Abstract Full Text PDF PubMed Google Scholar, 4Miller M.J. Gennis R.B. J. Biol. Chem. 1983; 258: 9159-9165Abstract Full Text PDF PubMed Google Scholar, 7Lorence R.M. Koland J.G. Gennis R.B. Biochemistry. 1986; 25: 2314-2321Crossref PubMed Scopus (82) Google Scholar).TABLE I Open table in a new tab Optical SpectroscopyThe optical absorption spectral change upon addition of cyanide to the air-oxidized O2-bound enzyme was examined. The 647 nm band that is known to originate from the cytochrome d (Fe2+)-O2 species (10Kahlow M.A. Loehr T.M. Zuberi T.M. Gennis R.B. J. Am. Chem. Soc. 1993; 115: 5845-5846Crossref Scopus (45) Google Scholar) disappeared in a time-dependent manner (Fig. 1), as previously reported(26Poole R.K. Kumar C. Salmon I. Chance B. J. Gen. Microbiol. 1983; 129: 1335-1344PubMed Google Scholar, 27Lorence R.M. Gennis R.B. J. Biol. Chem. 1989; 264: 7135-7140Abstract Full Text PDF PubMed Google Scholar). Simultaneously a Soret band spectral change (a peak at 431 nm and a trough at 411 nm in the air-oxidized cyanide-treated minus air-oxidized O2-bound difference spectrum) was observed (Fig. 1)(28Krasnoselskaya I. Arutjunjan A.M. Smirnova I. Gennis R. Konstantinov A.A. FEBS Lett. 1993; 327: 279-283Crossref PubMed Scopus (35) Google Scholar). Exposure of the air-oxidized O2-bound sample to a CO atmosphere caused a shift of the 647 nm peak to 635 nm, consistent with the formation of the cytochrome d (Fe2+)-CO species(3Kita K. Konishi K. Anraku Y. J. Biol. Chem. 1984; 259: 3375-3381Abstract Full Text PDF PubMed Google Scholar, 4Miller M.J. Gennis R.B. J. Biol. Chem. 1983; 258: 9159-9165Abstract Full Text PDF PubMed Google Scholar, 16Tsubaki M. Uno T. Hori H. Mogi T. Nishimura Y. Anraku Y. FEBS Lett. 1993; 335: 13-17Crossref PubMed Scopus (15) Google Scholar). The addition of cyanide to this air-oxidized CO-bound enzyme also caused the characteristic 635 nm band to disappear. The resulting species in both cases had a very broad absorption band around 630 nm, indicative of the oxidation of cytochrome d. Pretreatment of the air-oxidized O2-bound (or air-oxidized CO-bound) enzyme with cyanide retards the appearance of the reduced cytochrome d absorption band at 629 nm upon the addition of Na2S2O4 (spectra not shown), consistent with the previous results(29Kauffman H.F. van Gelder B.F. Biochim. Biophys. Acta. 1973; 314: 276-283Crossref PubMed Scopus (24) Google Scholar). On the other hand, the addition of cyanide (10 mM) to the fully reduced (with Na2S2O4) form of bd-type ubiquinol oxidase did not affect the visible absorption spectrum (data not shown)(29Kauffman H.F. van Gelder B.F. Biochim. Biophys. Acta. 1973; 314: 276-283Crossref PubMed Scopus (24) Google Scholar, 30Pudek M.R. Bragg P.D. Arch. Biochem. Biophys. 1974; 164: 682-693Crossref PubMed Scopus (57) Google Scholar).FT-IR SpectroscopyJust after the addition of cyanide (5 mM) to the air-oxidized O2-bound enzyme, there was no infrared band assignable to a bound cyanide species, except for a band of free H12C14N at 2093 cm-(31Yoshikawa S. O'Keeffe D.H. Caughey W.S. J. Biol. Chem. 1985; 260: 3518-3528Abstract Full Text PDF PubMed Google Scholar). A weak feature at 2161 cm- developed in a time-dependent manner (spectra not shown). This band showed cyanide isotope-sensitive shifts to 2129 cm- (12C15N), to 2114 cm- (13C14N), and to 2082 cm- (13C15N). The identical cyanide band developed, but more quickly, upon the addition of cyanide (5 mM) to the air-oxidized CO-bound enzyme, with a concomitant disappearance of the cytochrome d(Fe2+)-C-O stretching band at 1983.4 cm- (Fig. 2, A and B). The addition of Na2S2O4 to the cyanide-pretreated enzyme in the CO atmosphere reversed the spectral change. The cytochrome d(Fe2+)-C-O stretching band at 1980.7 cm- developed in a time-dependent manner with a concomitant decrease of the 2161 cm- band intensity (Fig. 2, C, D, and E). A shoulder around 1975 cm- was noticed in the early stage of the spectral change (Fig. 2C), and a weak C-N infrared band at 2138 cm- was observed during the spectral change (Fig. 2C). Pretreatment of the air-oxidized O2-bound enzyme with Na2S2O4 and CO before the addition of cyanide produced only an infrared band of the heme d(Fe2+)-bound C-O stretching mode at 1980.7 cm-(16Tsubaki M. Uno T. Hori H. Mogi T. Nishimura Y. Anraku Y. FEBS Lett. 1993; 335: 13-17Crossref PubMed Scopus (15) Google Scholar). The 2161 cm- species was not observed at all under the fully reduced conditions.Figure 2FT-IR spectra of bd-type ubiquinol oxidase in the C-O and C-N stretching vibration region. The air-oxidized O2-bound enzyme was exposed first to carbon monoxide (12C16O) atmosphere (A); then cyanide (12C14N; 5 mM) was added anaerobically to the air-oxidized CO-exposed enzyme, (B, taken 40 min after the addition of cyanide). Then Na2S2O4 was added anaerobically to the air-oxidized CO-exposed cyanide-treated enzyme, and the development of the 1980.7 cm- band and decay of the 2161 cm- band were recorded (C, D, and E; taken just after, 100 min after, and 16 h after the addition of Na2S2O4, respectively). Sample concentration was 0.60 mM (1.21 mM in heme B concentration). The band at 2093 cm- is due to free H12C14N.View Large Image Figure ViewerDownload Hi-res image Download (PPT)EPR SpectroscopyThe addition of cyanide (5 mM) to the air-oxidized O2-bound enzyme just before the EPR measurement caused changes in the spectral line shape of the g = 6 high spin signal region (spectra not shown). The line shapes of both the rhombic and axial components are altered, with the axial component being slightly broader and the rhombic signal being further distorted. These observations are essentially consistent with those reported for the membrane-bound bd-type ubiquinol oxidase(32Rothery R.A. Ingledew W.J. Biochem. J. 1989; 261: 437-443Crossref PubMed Scopus (38) Google Scholar). However, the intensity of the high spin signals did not change appreciably compared with that of the air-oxidized O2-bound state before addition of cyanide. Prolonged incubation of the enzyme with cyanide caused diminution of the g = 6 rhombic signal, whereas the axial high spin signal was little changed during the incubation (spectra not shown).Cyanide also caused disappearance of ferric low spin signals at gz = 2.46, gy = 2.32 (Fig. 3, A and B), and gx = 1.83 (not shown) at 15 K. Those were assigned to a minor form of ferric cytochrome d(9Hata A. Kirino Y. Matsuura K. Itoh S. Hiyama T. Konishi K. Kita K. Anraku Y. Biochim. Biophys. Acta. 1985; 810: 62-72Crossref PubMed Scopus (44) Google Scholar). Concomitantly, a derivative-shaped EPR signal at g = 3.15 developed (Fig. 3B) together with another low spin signal at g = 2.82. Prolonged incubation of the sample at 4°C in the dark caused an increase in intensity of these EPR signals (Fig. 3C). The EPR signal at g = 3.15 is distinct from the g = 3.3 signal previously assigned as ferric low spin cytochrome b558(32Rothery R.A. Ingledew W.J. Biochem. J. 1989; 261: 437-443Crossref PubMed Scopus (38) Google Scholar, 33Meinhardt S.W. Gennis R.B. Ohnishi T. Biochim. Biophys. Acta. 1989; 975: 175-184Crossref PubMed Scopus (39) Google Scholar, 34Spinner F. Chessman M.R. Thomson A.J. Kaysser T. Gennis R.B. Peng Q. Peterson J. Biochem. J. 1995; 308: 641-644Crossref PubMed Scopus (47) Google Scholar), although both signals overlapped.Figure 3Effects of cyanide addition on the EPR spectra of bd-type ubiquinol oxidase. Shown are: A, the air-oxidized O2-bound state; B, 10 min after addition of cyanide (5 mM); C, 1 week after addition of cyanide (5 mM); and D, just after addition of sodium dithionite to C. The conditions were: temperature, 8 K; modulation frequency, 5 Gauss; power, 5 mW; sample concentration, 0.179 mM (0.358 mM in heme B concentration).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Anaerobic addition of Na2S2O4 to this cyanide-pretreated form caused a rapid disappearance of the high spin signals together with the low spin signal at g = 2.82. The derivative-shaped EPR signal at g = 3.15 persisted and a transient EPR species with gz = 2.96 appeared (Fig. 3D). The gz = 2.96 signal can be ascribed to a cyanide adduct of cytochrome d(Fe3+)(32Rothery R.A. Ingledew W.J. Biochem. J. 1989; 261: 437-443Crossref PubMed Scopus (38) Google Scholar, 35Jünemann S. Wrigglesworth J.M. J. Biol. Chem. 1995; 270: 16213-16220Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Prolonged incubation of the Na2S2O4-treated sample on ice removed the derivative-shaped EPR signal and the transient EPR signal completely.DISCUSSIONNumber of Metal Centers in bd-type Ubiquinol OxidaseThe heme B and iron contents of the purified enzyme suggest that there are only three cytochromes present. Thus, we conclude that bd-type ubiquinol oxidase is a cytochrome bbd-type enzyme as reported previously(4Miller M.J. Gennis R.B. J. Biol. Chem. 1983; 258: 9159-9165Abstract Full Text PDF PubMed Google Scholar, 6Koland J.G. Miller M.J. Gennis R.B. Biochemistry. 1984; 23: 1051-1056Crossref Scopus (48) Google Scholar, 7Lorence R.M. Koland J.G. Gennis R.B. Biochemistry. 1986; 25: 2314-2321Crossref PubMed Scopus (82) Google Scholar). Assuming this structure, we recalculated the extinction coefficients for various wavelength pairs (Table 1). ( 2Recently, R. B. Gennis recalculated and obtained essentially the same extinction coefficients for the E. coli oxidase (personal communication).) The new set gave us reasonable estimation of the number of metal centers in the oxidase. We found that the preparations of Kita et al.(3Kita K. Konishi K. Anraku Y. J. Biol. Chem. 1984; 259: 3375-3381Abstract Full Text PDF PubMed Google Scholar), Konishi et al.(36Konishi K. Ouchi M. Kita K. Horikoshi I. J. Biochem. 1986; 99: 1227-1236Crossref PubMed Scopus (18) Google Scholar), and Lorence et al. (7Lorence R.M. Koland J.G. Gennis R.B. Biochemistry. 1986; 25: 2314-2321Crossref PubMed Scopus (82) Google Scholar) all contain two cytochromes b and one cytochrome d in the oxidase (i.e. 2:1.05, 2:0.76, 2:1.08 (b558:b595:d = 1:1.02:1.09), respectively).FT-IR SpectraOnly one bound cyanide infrared band (2161 cm-) was observed upon the addition of cyanide to the air-oxidized O2-bound enzyme. The increase of the band intensity was in parallel to the intensity decrease of the optical absorption band at 647 nm (Fig. 1). The same infrared band was formed upon the addition of cyanide to the air-oxidized CO-bound enzyme with concomitant disappearances of the 1983.4 cm- C-O infrared band and the 635 nm band of cytochrome d(Fe2+)-CO (Fig. 2). Thus, it is quite certain that cyanide binds at the cytochrome d(Fe3+) center. The frequency is in the region of the so-called "bridging cyanide" bound to metals in the oxidized state (i.e. 2181 ∼2141 cm-)(22Tsubaki M. Mogi T. Anraku Y. Hori H. Biochemistry. 1993; 32: 6065-6072Crossref PubMed Scopus (81) Google Scholar, 37Tsubaki M. Biochemistry. 1993; 32: 164-173Crossref PubMed Scopus (46) Google Scholar, 38Scott M.J. Holm R.H. J. Am. Chem. Soc. 1994; 116: 11357-11367Crossref Scopus (118) Google Scholar). Therefore, it is very likely that the 2161 cm- band is due to a species having a cyanide-bridging structure between heme d(Fe3+) and one of other two ferric heme irons.EPR SpectraThe postulated cyanide-bridging species is expected to be either EPR-inactive or to have an unusual EPR character because the spin-spin coupling depends on the relative geometry and the spin state of the participating metal centers. The derivative-shaped EPR signal at g = 3.15 is likely derived from the cyanide-bridging species (Fig. 3). The ferric low spin EPR signals at gz = 2.46, gy = 2.32, and gx = 1.83 that are assignable to a minor form of ferric cytochrome d(9Hata A. Kirino Y. Matsuura K. Itoh S. Hiyama T. Konishi K. Kita K. Anraku Y. Biochim. Biophys. Acta. 1985; 810: 62-72Crossref PubMed Scopus (44) Google Scholar) seem at least partially responsible for the appearance of the peculiar signal at g = 3.15 and the low spin signal at g = 2.82, because these EPR signals appeared just after the addition of cyanide. However, a major part of the g = 3.15 species developed as the oxidation of the oxygenated form of cytochrome d(Fe2+) proceeded after the addition of cyanide.On the other hand, the g = 6 high spin rhombic EPR signal diminished appreciably upon prolonged incubation with cyanide, whereas the axial high spin signal changed little during the incubation. Although the assignment for the g = 6 high spin axial signal ( 3Although the g = 3.3 signal has been assigned as the gz component of the ferric low spin species of cytochrome b558 in the air-oxidized state(32Rothery R.A. Ingledew W.J. Biochem. J. 1989; 261: 437-443Crossref PubMed Scopus (38) Google Scholar, 33Meinhardt S.W. Gennis R.B. Ohnishi T. Biochim. Biophys. Acta. 1989; 975: 175-184Crossref PubMed Scopus (39) Google Scholar, 34Spinner F. Chessman M.R. Thomson A.J. Kaysser T. Gennis R.B. Peng Q. Peterson J. Biochem. J. 1995; 308: 641-644Crossref PubMed Scopus (47) Google Scholar), we consider this signal as a minor form of cytochrome b558. The g = 3.3 signal seems to be too weak to account for the whole cytochrome b558 content. We suggest that a major population of cytochrome b558 in the air-oxidized O2-bound form adopts a high spin state that exhibits g = 6 axial EPR signal. This interpretation was confirmed recently by resonance Raman measurements of the air-oxidized O2-bound bd-type ubiquinol oxidase (S. Hirota, T. Mogi, Y. Anraku, and T. Kitagawa, unpublished observations). They could not observe any typical low spin marker bands in the higher frequency region with various excitation wavelengths around the Soret region.) is still controversial, there seems to be a consensus that the g = 6 high spin rhombic signal is due to cytochrome b595(32Rothery R.A. Ingledew W.J. Biochem. J. 1989; 261: 437-443Crossref PubMed Scopus (38) Google Scholar, 33Meinhardt S.W. Gennis R.B. Ohnishi T. Biochim. Biophys. Acta. 1989; 975: 175-184Crossref PubMed Scopus (39) Google Scholar, 39Hata-Tanaka A. Matsuura K. Itoh S. Anraku Y. Biochim. Biophys. Acta. 1987; 893: 289-295Crossref PubMed Scopus (41) Google Scholar). From these pieces of evidence we conclude that the cyanide-bridging structure at the active site is a kind of "heme d(Fe3+)-C=N-heme b595(Fe3+)."The nature of the EPR signal at g = 2.82 is not clear because the intensity of this signal was preparation-dependent. This may be due to a minor form of heme d(Fe3+)-CN derived from the cyanide-bridging species in which the bridging structure is partially destroyed upon freezing. This scenario is consistent with the observation of only one C-N stretching infrared band (i.e. the 2161 cm- band) at room temperature. However, other possibilities (e.g. heme b595(Fe3+)-CN or heme b595(Fe3+)-NC species) cannot be completely excluded.The addition of Na2S2O4 to the preformed cyanide complex produced a new, but transient, low spin heme species having with the gz = 2.96 EPR signal (Fig. 3D) and the 2138 cm- cyanide infrared band (Fig. 2). This species is assignable to heme d(Fe3+)-CN unambiguously(32Rothery R.A. Ingledew W.J. Biochem. J. 1989; 261: 437-443Crossref PubMed Scopus (38) Google Scholar, 35Jünemann S. Wrigglesworth J.M. J. Biol. Chem. 1995; 270: 16213-16220Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The observed spectral changes are most likely due to a breakage of the cyanide-bridging structure caused by the reduction of cytochrome b595 heme itself or by the conformational change around the binuclear center upon reduction of cytochrome b558 heme. Such influence of the redox states of other metal centers on the heme d has been observed for the bound C-O stretching frequencies, i.e. the C-O band appears at 1980.7 cm- in the fully reduced state, whereas it is at 1983.4 cm- in the partially reduced state (Fig. 2)(16Tsubaki M. Uno T. Hori H. Mogi T. Nishimura Y. Anraku Y. FEBS Lett. 1993; 335: 13-17Crossref PubMed Scopus (15) Google Scholar). These spectral characteristics are indicative of the heme-heme interactions and have some similarities to those of the heme-copper oxidase(22Tsubaki M. Mogi T. Anraku Y. Hori H. Biochemistry. 1993; 32: 6065-6072Crossref PubMed Scopus (81) Google Scholar, 37Tsubaki M. Biochemistry. 1993; 32: 164-173Crossref PubMed Scopus (46) Google Scholar, 40Yoshikawa S. Caughey W.S. J. Biol. Chem. 1982; 257: 412-420Abstract Full Text PDF PubMed Google Scholar).The lower gz value (2.96 for the partially reduced state and 2.82 for the air-oxidized state) and the higher C-N stretching frequency (2138 cm-) of the heme d(Fe3+)-CN moiety compared with those of corresponding species of the heme-copper oxidase (gz = 3.24 (41Ingledew W.J. Horrocks J. Salerno J.C. Eur. J. Biochem. 1993; 212: 657-664Crossref PubMed Scopus (16) Google Scholar) and 2123 cm( 4M. Tsubaki, H. Hori, T. Mogi, and Y. Anraku, unpublished results.) for the E. coli bo-type ubiquinol oxidase and gz = 3.58 (42Johnson M.K. Eglinton D.G. Gooding P.E. Greenwood C. Thomson A.J. Biochem. J. 1981; 193: 699-708Crossref PubMed Scopus (32) Google Scholar) and 2132 cm(37Tsubaki M. Biochemistry. 1993; 32: 164-173Crossref PubMed Scopus (46) Google Scholar) for bovine cytochrome c oxidase) might be ascribed, however, mainly to the chlorin macrocycle of heme d. It was reported that cyanide adducts of ferric hemoproteins containing chlorin macrocycle exhibited a gz value as low as 2.6(43Peng Q. Peterson J. FEBS Lett. 1994; 356: 159-161Crossref PubMed Scopus (4) Google Scholar). It is also known that presence of electron-withdrawing groups (such as hydroxyl groups) on iron porphyrin macrocycle leads to an increase of the bound C-N stretching vibration(31Yoshikawa S. O'Keeffe D.H. Caughey W.S. J. Biol. Chem. 1985; 260: 3518-3528Abstract Full Text PDF PubMed Google Scholar).Structure of the Heme-Heme Binuclear Metal CenterHill et al. (14Hill J.J. Alben J.O. Gennis R.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5863-5867Crossref PubMed Scopus (108) Google Scholar, 15Hill J.J. Gennis R.B. Alben J.O. Biophys. J. 1994; 66 (abstr.): 367Google Scholar) claimed that there is a heme d-heme b595 binuclear center at the catalytic site of bd-type ubiquinol oxidase on the basis of the low temperature FT-IR study. Heme b595 traps the photolyzed CO from cytochrome d and exhibits the 1974 cm- band at cryogenic temperature(14Hill J.J. Alben J.O. Gennis R.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5863-5867Crossref PubMed Scopus (108) Google Scholar). Later, Krasnoselskaya et al.(28Krasnoselskaya I. Arutjunjan A.M. Smirnova I. Gennis R. Konstantinov A.A. FEBS Lett. 1993; 327: 279-283Crossref PubMed Scopus (35) Google Scholar) proposed that cyanide reacts with the air-oxidized O2-bound bd-type ubiquinol oxidase to cause a decomposition of the cytochrome d-O2 complex and an oxidation of heme d resulting in a formation of a cytochrome d(Fe3+)-CN species. As one possibility, they proposed a structure like "heme d(Fe3+)-C=N-heme b595(Fe3+)." This is consistent with our present conclusion.We should consider, at this point, a possible structure of the heme-heme binuclear center to obtain detailed insight into the dioxygen reduction mechanism of bd-type quinol oxidase. In the cyanide-bridging structure, the C atom of the cyanide is likely bound to the Fe atom of heme d with the Fe-C bond being perpendicular to the heme plane (with the Fe-C and C=N bond lengths as 1.9 and 1.1 Å, respectively(38Scott M.J. Holm R.H. J. Am. Chem. Soc. 1994; 116: 11357-11367Crossref Scopus (118) Google Scholar)). The Fe(b595)-NC distance and Fe(b595)-N-C angle would vary markedly with the relative distance and orientation of the two metal centers (38Scott M.J. Holm R.H. J. Am. Chem. Soc. 1994; 116: 11357-11367Crossref Scopus (118) Google Scholar). The orientations of heme d and heme b595 have been determined by EPR studies on oriented multilayer preparations of cytoplasmic membrane fragments (44Ingledew W.J. Rothery R.A. Gennis R.B. Salerno J.C. Biochem. J. 1992; 282: 255-259Crossref PubMed Scopus (19) Google Scholar). Both ferric high and low spin heme d are oriented with their heme planes perpendicular to the membrane plane. Ferric high spin heme b595 is oriented with its heme plane at approximately 55° to the membrane plane. Thus, heme d and heme b595 planes are facing each other with 35° orientation. Assuming the Fe(b595)-N bond to be perpendicular to the heme plane with the bond length of 2.2 Å(38Scott M.J. Holm R.H. J. Am. Chem. Soc. 1994; 116: 11357-11367Crossref Scopus (118) Google Scholar), the distance of the two metal center was calculated as 5.1 Å. In this case, peripheral group(s) of the two hemes must be in a close contact each other, the access of an amino acid residue(s) into the ligand binding pocket (i.e. the space between the two metal centers) must be highly restricted, and the ligand-binding pocket itself would be very hydrophobic. The hydrophobic nature of the ligand-binding pocket is consistent with the unusual stability of the oxygenated species as well as the oxoferryl (Fe4+=O) adduct(10Kahlow M.A. Loehr T.M. Zuberi T.M. Gennis R.B. J. Am. Chem. Soc. 1993; 115: 5845-5846Crossref Scopus (45) Google Scholar, 11Kahlow M.A. Zuberi T.M. Gennis R.B. Loehr T.M. Biochemistry. 1991; 30: 11485-11489Crossref PubMed Scopus (64) Google Scholar). This might be one of the reasons why bd-type ubiquinol oxidase is more resistant to anionic inhibitors such as cyanide and azide than bo-type ubiquinol oxidase(2Poole R.K. Biochim. Biophys. Acta. 1983; 726: 205-243Crossref PubMed Scopus (206) Google Scholar, 3Kita K. Konishi K. Anraku Y. J. Biol. Chem. 1984; 259: 3375-3381Abstract Full Text PDF PubMed Google Scholar). INTRODUCTIONEscherichia coli has two terminal ubiquinol oxidases in the aerobic electron transfer chain: bo-type ubiquinol oxidase, which is expressed under high oxygen tension, and bd-type ubiquinol oxidase, which predominates under low oxygen tension(1Anraku Y. Annu. Rev. Biochem. 1988; 57: 101-132Crossref PubMed Scopus (170) Google Scholar, 2Poole R.K. Biochim. Biophys. Acta. 1983; 726: 205-243Crossref PubMed Scopus (206) Google Scholar). These oxidases are structurally unrelated, but both catalyze the two-electron oxidation of ubiquinol-8 and the four-electron reduction of dioxygen to produce water(1Anraku Y. Annu. Rev. Biochem. 1988; 57: 101-132Crossref PubMed Scopus (170) Google Scholar, 2Poole R.K. Biochim. Biophys. Acta. 1983; 726: 205-243Crossref PubMed Scopus (206) Google Scholar). The E. coli bd-type ubiquinol oxidase has been isolated and was found to consist of two polypeptide chains: subunit I (58 kDa) and subunit II (43 kDa)(3Kita K. Konishi K. Anraku Y. J. Biol. Chem. 1984; 259: 3375-3381Abstract Full Text PDF PubMed Google Scholar, 4Miller M.J. Gennis R.B. J. Biol. Chem. 1983; 258: 9159-9165Abstract Full Text PDF PubMed Google Scholar). The cydAB genes coding for both polypeptides have been cloned and sequenced(5Green G.N. Fang H. Lin R.-J. Newton G. Mather M. Georgiou C.D. Gennis R.B. J. Biol. Chem. 1988; 263: 13138-13143Abstract Full Text PDF PubMed Google Scholar). Within this enzyme, it has been claimed, there are three types of cytochrome heme species based on the optical spectra: cytochrome d, cytochrome b595, and cytochrome b558(6Koland J.G. Miller M.J. Gennis R.B. Biochemistry. 1984; 23: 1051-1056Crossref Scopus (48) Google Scholar, 7Lorence R.M. Koland J.G. Gennis R.B. Biochemistry. 1986; 25: 2314-2321Crossref PubMed Scopus (82) Google Scholar). Cytochrome d has a chlorin chromophore (heme D) (8Timkovich R. Cork M.S. Gennis R.B. Johnson P.Y. J. Am. Chem. Soc. 1985; 107: 6069-6075Crossref Scopus (102) Google Scholar) exhibiting a characteristic absorption maximum at 628 nm in the fully reduced state and is a primary binding site for exogenous ligands(3Kita K. Konishi K. Anraku Y. J. Biol. Chem. 1984; 259: 3375-3381Abstract Full Text PDF PubMed Google Scholar, 4Miller M.J. Gennis R.B. J. Biol. Chem. 1983; 258: 9159-9165Abstract Full Text PDF PubMed Google Scholar, 9Hata A. Kirino Y. Matsuura K. Itoh S. Hiyama T. Konishi K. Kita K. Anraku Y. Biochim. Biophys. Acta. 1985; 810: 62-72Crossref PubMed Scopus (44) Google Scholar). The dioxygen molecule forms a very stable adduct with ferrous cytochrome d showing its Fe2+-O2 stretching vibration at 568 cm- (essentially identical to that of myoglobin)(10Kahlow M.A. Loehr T.M. Zuberi T.M. Gennis R.B. J. Am. Chem. Soc. 1993; 115: 5845-5846Crossref Scopus (45) Google Scholar), and thus the enzyme in the air-oxidized state is actually an oxygenated form. The cytochrome d moiety also forms a remarkably stable oxoferryl (Fe4+=O) adduct(11Kahlow M.A. Zuberi T.M. Gennis R.B. Loehr T.M. Biochemistry. 1991; 30: 11485-11489Crossref PubMed Scopus (64) Google Scholar). Subunit I contains cytochrome b558 that shows its α and β peaks at 562 and 532 nm, respectively, in the reduced state (12Green G.N. Kranz R.G. Lorence R.M. Gennis R.B. J. Biol. Chem. 1984; 259: 7994-7997Abstract Full Text PDF PubMed Google Scholar) and most likely forms the ubiquinol-8 binding site(13Yang F.-D. Yu L. Yu C.-A. Lorence R.M. Gennis R.B. J. Biol. Chem. 1986; 261: 14987-14990Abstract Full Text PDF PubMed Google Scholar). Cytochrome b595 is an unusual b-type cytochrome. It was suggested that the optical spectrum of this cytochrome is very similar to that of high spin cytochrome c peroxidase, and its α and β bands are at 595 and 562 nm, respectively, in the reduced minus oxidized difference spectrum(7Lorence R.M. Koland J.G. Gennis R.B. Biochemistry. 1986; 25: 2314-2321Crossref PubMed Scopus (82) Google Scholar). Recent low temperature CO photolysis experiments monitored by the infrared C-O stretching absorption suggest that cytochrome d and cytochrome b595 form a binuclear center(14Hill J.J. Alben J.O. Gennis R.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5863-5867Crossref PubMed Scopus (108) Google Scholar, 15Hill J.J. Gennis R.B. Alben J.O. Biophys. J. 1994; 66 (abstr.): 367Google Scholar).In a previous study we analyzed the purified bd-type ubiquinol oxidase by resonance Raman, FT-IR, ( 1The abbreviations used are: FT-IRFourier-transform infraredEPRelectron paramagnetic resonance.) and EPR spectroscopies (16Tsubaki M. Uno T. Hori H. Mogi T. Nishimura Y. Anraku Y. FEBS Lett. 1993; 335: 13-17Crossref PubMed Scopus (15) Google Scholar). We found that heme d-bound C-O and Fe2+-CO stretching frequencies were at 1980.7 and 471 cm-, respectively, in the fully reduced state. These values differ considerably from those of the heme-copper respiratory oxidases and the oxygen-carrying hemoproteins, both groups possessing a His residue as the heme axial ligand(17Tsubaki M. Srivastava R.B. Yu N.-T. Biochemistry. 1982; 21: 1132-1140Crossref PubMed Scopus (181) Google Scholar, 18Argade P.V. Ching Y. Rousseau D.L. Science. 1984; 225: 329-331Crossref PubMed Scopus (65) Google Scholar, 19Uno T. Nishimura Y. Tsuboi M. Kita K. Anraku Y. J. Biol. Chem. 1985; 260: 6755-6760Abstract Full Text PDF PubMed Google Scholar, 20Uno T. Mogi T. Tsubaki M. Nishimura Y. Anraku Y. J. Biol. Chem. 1994; 269: 11912-11920Abstract Full Text PDF PubMed Google Scholar). EPR analyses on the air-oxidized O2-bound and Na2S2O4-reduced nitric oxide-bound forms revealed that there is no superhyperfine structure originating from the heme axial 14N ligand in the central resonance of the nitric oxide EPR signals(16Tsubaki M. Uno T. Hori H. Mogi T. Nishimura Y. Anraku Y. FEBS Lett. 1993; 335: 13-17Crossref PubMed Scopus (15) Google Scholar). These results suggest strongly that the heme d axial ligand of bd-type ubiquinol oxidase is either a His residue in an anomalous condition or some other residue making the molecular structure around the oxygen-binding site different from those of the heme-copper respiratory oxidases. Indeed, electron nuclear double resonance spectroscopy has shown that the axial ligand to heme d is most probably not a histidine or other strong nitrogenous ligand(21Jiang F.S. Zuberi T.M. Cornelius J.B. Clarkson R.B. Gennis R.B. Belford R.L. J. Am. Chem. Soc. 1993; 115: 10293-10299Crossref Scopus (1) Google Scholar). In the present study we extended our investigation on the redox centers of the E. coli bd-type ubiquinol oxidase using cyanide as a monitoring probe.