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Spectral Properties of Bacterial Nitric-oxide Reductase

2002; Elsevier BV; Volume: 277; Issue: 23 Linguagem: Inglês

10.1074/jbc.m112202200

1083-351X

Sarah J. Field, Louise Prior, María Dolores Roldán, Myles R. Cheesman, Andrew J. Thomson, Stephen Spiro, Julea N. Butt, Nicholas J. Watmough, David J. Richardson,

Hemoglobin structure and function

Bacterial nitric-oxide reductase catalyzes the two electron reduction of nitric oxide to nitrous oxide. In the oxidized form the active site non-heme FeB and high spin heme b3 are μ-oxo bridged. The heme b3 has a ligand-to-metal charge transfer band centered at 595 nm, which is insensitive to pH over the range of 6.0–8.5. Partial reduction of nitric-oxide reductase yields a three electron-reduced state where only the heme b3 remains oxidized. This results in a shift of the heme b3 charge transfer band λmax to longer wavelengths. At pH 6.0 the charge transfer band λmax is 605 nm, whereas at pH 8.5 it is 635 nm. At pH 6.5 and 7.5 the nitric-oxide reductase ferric heme b3 population is a mixture of both 605- and 635-nm forms. Magnetic circular dichroism spectroscopy suggests that at all pH values examined the proximal ligand to the ferric heme b3 in the three electron-reduced form is histidine. At pH 8.5 the distal ligand is hydroxide, whereas at pH 6.0, when the enzyme is most active, it is water. Bacterial nitric-oxide reductase catalyzes the two electron reduction of nitric oxide to nitrous oxide. In the oxidized form the active site non-heme FeB and high spin heme b3 are μ-oxo bridged. The heme b3 has a ligand-to-metal charge transfer band centered at 595 nm, which is insensitive to pH over the range of 6.0–8.5. Partial reduction of nitric-oxide reductase yields a three electron-reduced state where only the heme b3 remains oxidized. This results in a shift of the heme b3 charge transfer band λmax to longer wavelengths. At pH 6.0 the charge transfer band λmax is 605 nm, whereas at pH 8.5 it is 635 nm. At pH 6.5 and 7.5 the nitric-oxide reductase ferric heme b3 population is a mixture of both 605- and 635-nm forms. Magnetic circular dichroism spectroscopy suggests that at all pH values examined the proximal ligand to the ferric heme b3 in the three electron-reduced form is histidine. At pH 8.5 the distal ligand is hydroxide, whereas at pH 6.0, when the enzyme is most active, it is water. Bacterial nitric-oxide reductases (NOR) 1The abbreviations used are: NORnitric-oxide reductaseCTcharge transferRT-MCDroom temperature magnetic circular dichroismBTPbis-Tris propanePMSphenazine methosulfatePESphenazine ethosulfate.1The abbreviations used are: NORnitric-oxide reductaseCTcharge transferRT-MCDroom temperature magnetic circular dichroismBTPbis-Tris propanePMSphenazine methosulfatePESphenazine ethosulfate. catalyze shown in the following Reaction 1.2NO+2e−+2H+→N2O+H2OREACTION 1This reaction serves either as a key step in the pathway of denitrification that uses N-oxyanions and N-oxides as respiratory electron acceptors or as a way of removing cytotoxic NO (1Watmough N.J. Butland G. Cheesman M.R. Moir J.W.B. Richardson D.J. Spiro S. Biochim. Biophys. Acta. 1999; 1411: 456-474Crossref PubMed Scopus (118) Google Scholar). The capacity for NO reduction is now recognized in a phylogenetically diverse range of bacteria, which includes soil denitrifying bacteria such as Paracoccus denitrificans and pathogenic bacteria such as Neiserria meningitidis (2Hendricks J. Gohlke U. Saraste M. J. Bioenerg. Biomembr. 1998; 30: 15-24Crossref PubMed Scopus (60) Google Scholar, 3Cramm R. Pohlmann A. Friedrich B. FEBS Lett. 1999; 460: 6-10Crossref PubMed Scopus (73) Google Scholar, 4Hendricks J. Warne A. Gohlke U. Haltia T. Ludovici C. Lubben M. Saraste M. Biochemistry. 1998; 37: 13102-13109Crossref PubMed Scopus (116) Google Scholar). Three classes of NOR have been identified. The two-subunit (NorCB)-dependent class from P. denitrificans, Pseudomonas stutzeri, and Rhodobacter sphaeroides, which use cytochromes c or cupredoxins as an electron donor, the single subunit (NorB) quinol-oxidizing class from Ralstonia eutropha and N. meningitidis, and the CuA containing quinol and cytochrome c oxidizing enzyme of Bacillus azotoformans (2Hendricks J. Gohlke U. Saraste M. J. Bioenerg. Biomembr. 1998; 30: 15-24Crossref PubMed Scopus (60) Google Scholar, 3Cramm R. Pohlmann A. Friedrich B. FEBS Lett. 1999; 460: 6-10Crossref PubMed Scopus (73) Google Scholar, 5Suharti Strampraad M.J.F. Schröder I. de Vries S. Biochemistry. 2001; 40: 2632-2639Crossref PubMed Scopus (87) Google Scholar). nitric-oxide reductase charge transfer room temperature magnetic circular dichroism bis-Tris propane phenazine methosulfate phenazine ethosulfate. nitric-oxide reductase charge transfer room temperature magnetic circular dichroism bis-Tris propane phenazine methosulfate phenazine ethosulfate. Primary structure analysis, in combination with spectroscopic studies, has clearly established NORs as divergent members of the family of respiratory heme-copper oxidases. Characterization of P. denitrificans NorBC has established that the catalytic NorB subunit binds a bis-histidine-coordinated heme b that is functionally equivalent to heme a in cytochrome-c oxidase. It also binds a high spin heme b (termed b3) that is equivalent to heme a3 in cytochrome-c oxidase and heme o3 of Escherichia coli cytochrome-bo3 quinol oxidase. In cytochrome-c oxidase and quinol oxidases this heme is magnetically coupled to a copper ion (CuB) to form a dinuclear center, which is the site of oxygen binding and reduction. By contrast, in NOR this CuB is replaced with a non-heme iron, FeB (6Girsch P. de Vries S. Biochim. Biophys. Acta. 1997; 1318: 202-216Crossref PubMed Scopus (151) Google Scholar, 7Sukurai N. Sakurai T. Biochemistry. 1997; 36: 13809-13815Crossref PubMed Scopus (51) Google Scholar, 8Grönberg K.L.C. Roldàn M.D. Prior L. Butland G.P. Cheesman M.R. Richardson D.J. Spiro S. Thomson A.J. Watmough N.J. Biochemistry. 1999; 38: 13780-13786Crossref PubMed Scopus (99) Google Scholar), which is probably ligated by the three conserved histidine residues that serve as ligands to CuBin cytochrome-c oxidase. A fourth metal center, a covalently bound low spin heme c with histidine and methionine axial ligands, is bound by the NorC subunit. This site has no structural counterpart in cytochrome-c oxidase but is functionally equivalent to CuA in that it serves as a site of electron input for the respiratory complex. Recent NOR studies have addressed the nature of the catalytic site through site-directed mutagenesis (9Butland G. Spiro S. Watmough N.J. Richardson D.J. J. Bacteriol. 2001; 183: 189-199Crossref PubMed Scopus (96) Google Scholar), redox potentiometry (8Grönberg K.L.C. Roldàn M.D. Prior L. Butland G.P. Cheesman M.R. Richardson D.J. Spiro S. Thomson A.J. Watmough N.J. Biochemistry. 1999; 38: 13780-13786Crossref PubMed Scopus (99) Google Scholar), and ligand binding studies (6Girsch P. de Vries S. Biochim. Biophys. Acta. 1997; 1318: 202-216Crossref PubMed Scopus (151) Google Scholar). The redox potentiometry revealed that the high spin heme b3 of the dinuclear center had a surprisingly low midpoint redox potential (Em(pH 7.6) = +60 mV) (8Grönberg K.L.C. Roldàn M.D. Prior L. Butland G.P. Cheesman M.R. Richardson D.J. Spiro S. Thomson A.J. Watmough N.J. Biochemistry. 1999; 38: 13780-13786Crossref PubMed Scopus (99) Google Scholar), which imposes a large thermodynamic barrier to reduction by the low spin electron transferring heme c (Em(pH 7.6) = +310 mV) and heme b (Em(pH 7.6) = +345 mV). This may be a means by which reduction of the heme b3 is avoided to prevent forming a potentially dead-end ferrous heme-nitrosyl species with NO (8Grönberg K.L.C. Roldàn M.D. Prior L. Butland G.P. Cheesman M.R. Richardson D.J. Spiro S. Thomson A.J. Watmough N.J. Biochemistry. 1999; 38: 13780-13786Crossref PubMed Scopus (99) Google Scholar). It has also been noted that reduction of the FeB in the dinuclear center, which occurs when three electrons are introduced to the enzyme, results in a shift in the absorption maximum of the ligand to metal charge-transfer (CT) band associated with the high spin ferric heme b3 from ∼595 to 605 nm (8Grönberg K.L.C. Roldàn M.D. Prior L. Butland G.P. Cheesman M.R. Richardson D.J. Spiro S. Thomson A.J. Watmough N.J. Biochemistry. 1999; 38: 13780-13786Crossref PubMed Scopus (99) Google Scholar). Taken together with recent resonance Raman studies of NOR (10Moënne-Loccoz P. Richter O-M. H. Huang H-W. Wassser I.M. Ghiladi R.A. Karlin K.D. de Vries S. J. Am. Chem. Soc. 2000; 122: 9344-9345Crossref Scopus (84) Google Scholar), this observation can be accounted for by a change in the ligation from a μ-oxo bridged dinuclear center in which there is no proximal ligand to the ferric heme b3, to a form of the ferric heme b3 with a proximal histidine ligand and an anionic distal ligand (8Grönberg K.L.C. Roldàn M.D. Prior L. Butland G.P. Cheesman M.R. Richardson D.J. Spiro S. Thomson A.J. Watmough N.J. Biochemistry. 1999; 38: 13780-13786Crossref PubMed Scopus (99) Google Scholar). There is currently no agreement on a model for the catalytic cycle of NOR. However, it must involve the transfer of two protons and two electrons to the active site as these are required for the reduction of two NO molecules to N2O and H2O (Reaction 1). It is now generally agreed that these protons are moved to the active site from the periplasm (3Cramm R. Pohlmann A. Friedrich B. FEBS Lett. 1999; 460: 6-10Crossref PubMed Scopus (73) Google Scholar). In membranes this overall process is non-electrogenic because the electrons are also derived from donors located in the periplasm. A possible proton-conducting pathway that involves one or more conserved glutamate residues has emerged from site-specific mutagenesis of NOR (9Butland G. Spiro S. Watmough N.J. Richardson D.J. J. Bacteriol. 2001; 183: 189-199Crossref PubMed Scopus (96) Google Scholar). Two recent NOR studies have reported that the spectroscopic properties of the fully oxidized enzyme are not significantly affected by pH (10Moënne-Loccoz P. Richter O-M. H. Huang H-W. Wassser I.M. Ghiladi R.A. Karlin K.D. de Vries S. J. Am. Chem. Soc. 2000; 122: 9344-9345Crossref Scopus (84) Google Scholar,11Shiniji K. Sakurai N. Sakurai T. J. Inorg. Biochem. 2001; 83: 281-286Crossref PubMed Scopus (6) Google Scholar). These observations are perhaps surprising given the requirement of proton uptake to the dinuclear center for NO reduction. This work reports an electronic absorption and magnetic circular dichroism (MCD) spectroscopic study of NorCB from P. denitrificans at a range of pH and redox states. This has led to the identification of three spectrally distinct forms of the ferric heme b3, which may reflect μ-oxo bridged, hydroxide-bound, and water-bound species. The latter two species are only observed in the three electron-reduced enzyme and are pH-dependent. The results may help us to elucidate the catalytic cycle of NOR. The source of the NOR used in this study was P. denitrificans strain 93.11 (ΔctaDI, ΔctaDII qoxB::kan R) (12de Gier J.-W. L. Lübben M. Reijnders W.N.M. Tipker C.A. van Spanning R.J.M. Stouthamer A.H. van der Oost J. Mol. Microbiol. 1994; 13: 183-196Crossref PubMed Scopus (110) Google Scholar) grown in batch culture in minimal medium under anaerobic denitrifying conditions. The two-subunit form of the enzyme was purified essentially as described by Grönberg et al. (8Grönberg K.L.C. Roldàn M.D. Prior L. Butland G.P. Cheesman M.R. Richardson D.J. Spiro S. Thomson A.J. Watmough N.J. Biochemistry. 1999; 38: 13780-13786Crossref PubMed Scopus (99) Google Scholar). Electronic absorbance spectra were recorded either on an Aminco DW2000 spectrophotometer or a Hitachi U3000 spectrophotometer. Room temperature magnetic circular dichroism (RT-MCD) spectra were recorded on a Jasco J-500D circular dichrograph. An Oxford Instruments super-conducting solenoid with a 25-mm room temperature bore was used to generate magnetic fields of up to 6 tesla. MCD spectral intensities depend linearly on the magnetic field at room temperature and are expressed per unit magnetic field as Δε/H (m −1 cm−1tesla−1). Mediated equilibrium redox titrations of NOR were done at 20 °C in 20 mm bis-Tris propane (BTP) supplemented with 0.05% (w/v) dodecyl maltoside, 0.5 mm EDTA, and 340 mm NaCl and adjusted to the required pH. The methodology was essentially as that described by Dutton (13Dutton P.L. Methods Enzymol. 1978; 54: 411-435Crossref PubMed Scopus (724) Google Scholar). Dithionite was used as the reductant and potassium ferricyanide as the oxidant. The redox mediators, each at a final concentration of 10 μm, were phenazine methosulfate (PMS), phenazine ethosulfate (PES), 5-anthraquinone 2-sulfonate, 6-anthraquinone 2,6-disulfonate, and benzylviologen. A solution of saturated quinhydrone at pH 7 was used as a redox standard (E = +295 mV). All potentials quoted are with respect to the standard hydrogen electrode. Preparation of three electron-reduced NOR under the control of a potentiostat was achieved using a three-electrode cell configuration with a closed sample compartment thermostated at 4 °C. All manipulations were performed in an anaerobic chamber (N2atmosphere with O2 at less than 2 ppm). A 200-μl sample of 25 μm NOR in 20 mm BTP, 0.05% (w/v) dodecyl maltoside, 0.34 m NaCl, and 0.5 mm EDTA at the desired pH and supplemented with a mixture of redox mediators, which included ferricyanide, PES, PMS, and 2,6-dimethylbenzaquinone, was placed in a glassy carbon pot that provided the working electrode. A Ag/AgCl reference electrode and a platinum foil counter electrode contacted the solution through a Luggin tip and Vycor frit, respectively. The potential of the carbon pot was held at +150 mV with respect to the standard hydrogen electrode for 1 h, during which time the sample was stirred, and the current flowing from the cell fell to a negligible level. After this time the sample was withdrawn from the electrochemical cell and transferred to an anaerobic cell for MCD spectroscopy. Samples of P. denitrificans NOR were equilibrated at pH 6.5, 7.0, 7.5, 8.0, and 8.5 in BTP buffer. Samples were poised at a range of potentials between +400 and 0 mV and absorbance spectra were collected in the range of 500 to 700 nm. At each pH value the oxidized (∼+400 mV) spectrum showed the characteristic α/β band absorption features of low spin ferric hemes in the region 520–570 nm (Fig. 1). These features have previously been assigned to the histidine/methionine-ligated c-heme of the NorC subunit and the bis-histidine-ligated b heme of the NorB subunit (14Cheesman M.R. Zumft W.G. Thomson A.J. Biochemistry. 1998; 37: 3994-4000Crossref PubMed Scopus (68) Google Scholar). Each sample also exhibited a clearly resolved absorption shoulder at ∼595 nm. The λmax and intensity of this band was not significantly affected by the pH of the bulk phase. This 595-nm feature arises from a CT band associated with ferric high spin heme b3, which has no proximal ligand but has a distal μ-oxo bridge to the FeB (10Moënne-Loccoz P. Richter O-M. H. Huang H-W. Wassser I.M. Ghiladi R.A. Karlin K.D. de Vries S. J. Am. Chem. Soc. 2000; 122: 9344-9345Crossref Scopus (84) Google Scholar, 14Cheesman M.R. Zumft W.G. Thomson A.J. Biochemistry. 1998; 37: 3994-4000Crossref PubMed Scopus (68) Google Scholar). Complete reduction (E = ∼0 mV) of NOR at each pH led to the appearance of the intense absorption peaks at 550 and 560 nm that arise from reduction of the low spin heme c (550 nm) and heme b (560 nm) (Fig. 1). The heme b3 595 nm CT band disappears on reduction. Again, analysis of the reduced spectrum at each pH value revealed no substantial differences in the λmax and intensity of the absorption peaks. In an earlier study of NOR (8Grönberg K.L.C. Roldàn M.D. Prior L. Butland G.P. Cheesman M.R. Richardson D.J. Spiro S. Thomson A.J. Watmough N.J. Biochemistry. 1999; 38: 13780-13786Crossref PubMed Scopus (99) Google Scholar) carried out in Tris-HCl buffer at pH 7.6, it was demonstrated that lowering the potential of the NOR sample buffer from approximately +400 to +150 mV results in the reduction of the low spin hemes (heme b and heme c) and the non-heme iron (FeB). In the resulting three electron-reduced species the λmax of the heme b3 CT band shifts from 595 to ∼605 nm, and the extinction coefficient decreases. Thus, reduction of the FeB in the dinuclear center resulted in a change in the coordination environment of the heme b3. On the basis of MCD analysis and taking into account recent resonance Raman studies (10Moënne-Loccoz P. Richter O-M. H. Huang H-W. Wassser I.M. Ghiladi R.A. Karlin K.D. de Vries S. J. Am. Chem. Soc. 2000; 122: 9344-9345Crossref Scopus (84) Google Scholar), this coordination change is likely to be a rebinding of the proximal histidine and the breaking of the μ-oxo bridge to form a His/anion species. In the present study the three electron-reduced state of the enzyme has been investigated at a range of pH values in BTP buffer, which allows examination over a broad range of pH values and temperatures. Chemical poising of samples at +150 mV led to the reduction of the two low spin hemes and the FeB, whereas the high spin heme b3 remained oxidized. Analysis of these samples revealed that the absorption intensity of the red-shifted CT band is strongly affected by pH. The CT band is least intense at pH 6.5 and most intense at pH 8.5 (Fig. 1, A and D). This suggests that the form of the heme b3 present at pH 8.5 is different from that present at pH 6.5. Examination of (three electron-reduced) − (fully reduced) difference spectra (Fig. 2) shows that at pH 8.5 the CT band is positioned at 605 nm. At pH 7.5 the heme b3appears to in a mixed form, with one population exhibiting a 605-nm CT band and a second population exhibiting a 635-nm CT band. At pH 6.5 the 635-nm form dominates the spectrum. From these data, molar extinction coefficients of ε605–700 or ε700–635 as 3.66 mm −1 cm−1 and 1.18 mm −1 cm−1 were calculated for the 605 and 635-nm bands, respectively. These data suggest that at different pH values the ferric heme b3 in the three electron-reduced enzyme has a different distal ligand. High spin ferric hemes give two characteristic CT bands that produce derivative-shaped features in the MCD spectrum (15Brill A.S. Williams R.J.P. Biochem. J. 1961; 78: 246-253Crossref PubMed Google Scholar, 16Cheng J.C. Osborne G.A. Stephens P.J. Eton W.A. Nature. 1973; 241: 193-194Crossref Scopus (45) Google Scholar). The higher energy band is seen in the 600–700-nm region, and its precise wavelength is sensitive to the nature of the axial ligands (Table I). RT-MCD studies have been successfully used to assign the exogenous-bound distal ligand of histidine-ligated high spin ferric heme of E. coli cytochrome bo3 (30Watmough N.J. Cheesman M.R. Butler C.S. Little R.H. Greenwood C. Thomson A.J. J. Bioenerg. Biomembr. 1998; 30: 55-62Crossref PubMed Scopus (44) Google Scholar). Thus, this approach was exploited to determine the likely nature of the distal ligand to NOR heme b3 samples buffered at pH 6.0 and 8.5. Samples were poised electrochemically at +150 mV and examined by electronic absorption and RT-MCD spectroscopy. Electrochemical poising allowed the preparation in a small volume of the concentrated samples required for MCD analysis. The electronic absorption spectra clearly show that at pH 6.0 the heme b3 is in a pure 635-nm form and at pH 8.5 in a pure 605-nm form (Fig. 3 A, inset). The RT-MCD spectra show peaks in the α, β, and Soret regions dominated by signals from low spin ferrous heme. CT bands associated with the high spin ferric heme b3 are seen in the 600–650-nm region of the RT-MCD spectrum. Analysis of this region shows a band at 620 nm in the pH 8.5 sample, which is characteristic of high spin heme with a proximal histidine and a distal hydroxide (His/OH−). In the pH 6.0 sample this band is red-shifted to 640 nm, a position characteristic of His/H2O-ligated high spin ferric heme (Table I). This analysis is in good agreement with the UV-visible spectroscopic analysis of the three electron-reduced NOR generated by chemical reduction and confirms that the ligand change is not an artifact of reduction with sodium dithionite. Moreover, these data suggest that direct ligation of the heme b3 is dependent on both the redox state of the dinuclear center and on pH. In the oxidized form the heme b3 and non-heme FeB are bridged by a μ-oxo group. As the non-heme FeB is reduced, the heme b3 becomes ligated by hydroxide at high pH levels (∼8.5) and by water at low pH levels (∼6). Recent evidence from a study of carbon monoxide binding to the fully reduced (four electron-reduced) NOR suggests that these coordination states are retained by ferrous heme b3 (31Hendichs J.H.M. Prior L. Baker A.R. Thomson A.J. Saraste M. Watmough N.J. Biochemistry. 2001; 40: 13361-13369Crossref PubMed Scopus (39) Google Scholar).Table IRoom temperature absorption and MCD wavelengths for CT2 of high-spin ferric hemesLigand setProtein derivative1-aMb, myoglobin; HH, horse heart; SW, sperm whale; Lb, soybean leghemoglobin; HRP, horse radish peroxidase; c′, cytochrome c′.Absorption peakRT-MCD trough1-bIn multiheme proteins the positive part of this MCD feature is often obscured by adjacent low spin heme bands; the wavelength of the intensity minimum to longer wavelengths has been quoted.ReferencesnmnmHistidine/H2OHH & SW Mb, Lb626–631635–64617Seward H.E. Magneto-optical Spectroscopy of Hemoproteins. Ph.D. thesis. University of East Anglia, 1999Google Scholar, 18Yoshida S. Iizuka T. 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Acta. 1976; 427: 652-662Crossref PubMed Scopus (54) Google ScholarHistidine/F−HRP, SW & HH Mb,605–620621–62319Vickery L. Nozawa T. Sauer K. J. Am. Chem. Soc. 1976; 98: 343-350Crossref PubMed Scopus (158) Google Scholar, 22Dawson J.H. Holm R.H. Trudell J.R. Barth G. Linder R.E. Bunnenberg E. Djerassi C. Tang S.C. J. Am. Chem. Soc. 1976; 98: 3707-3709Crossref PubMed Scopus (183) Google Scholar, 24Nozawa T. Kobayashi N. Hatano M. Biochim. Biophys. Acta. 1976; 427: 652-662Crossref PubMed Scopus (54) Google Scholar, 25Springall J. Stillman M.J. Thomson A.J. Biochim. Biophys. Acta. 1976; 453: 494-501Crossref PubMed Scopus (29) Google ScholarHistidine/Tyr−SW Mb H64Y60061817Seward H.E. Magneto-optical Spectroscopy of Hemoproteins. Ph.D. thesis. University of East Anglia, 1999Google ScholarHistidine/RCOO−Mb + HCOO−, +H3COO−634–64526Dawson J.H. Dooly D.M. Lever A.B.P. Gray H.B. Physical Bioinorganic Chemistry Series, Iron Porphyrins, Part III. VCH Publishers, Inc., New York1989Google Scholar, 27Kobayashi N. Nozawa T. Hatano M. Biochim. Biophys. Acta. 1977; 493: 340-351Crossref PubMed Scopus (30) Google ScholarHistidine/−HRP, c′, BrCN-Mb638–645658–66317Seward H.E. Magneto-optical Spectroscopy of Hemoproteins. Ph.D. thesis. University of East Anglia, 1999Google Scholar, 24Nozawa T. Kobayashi N. Hatano M. Biochim. Biophys. Acta. 1976; 427: 652-662Crossref PubMed Scopus (54) Google Scholar, 28Monkara F. Bingham S.J. Fahmi F.H.A. McEwan A.G. Thomson A.J. Thurgood A.G.P. Moore G.R. Biochim. Biophys. Acta. 1992; 1100: 184-188Crossref PubMed Scopus (25) Google Scholar, 29Bracete A.M. Sono M. Dawson J.H. Biochim. Biophys. Acta. 1991; 1080: 264-270Crossref PubMed Scopus (31) Google ScholarHistidine/N3−SW & HH Mb∼630∼64019Vickery L. Nozawa T. Sauer K. J. Am. Chem. Soc. 1976; 98: 343-350Crossref PubMed Scopus (158) Google Scholar, 23Eglinton D.G. Gadsby P.M.A. Sievers G. Peterson J. Thomson A.J. Biochim. Biophys. Acta. 1983; 742: 648-658Crossref PubMed Scopus (27) Google Scholar1-a Mb, myoglobin; HH, horse heart; SW, sperm whale; Lb, soybean leghemoglobin; HRP, horse radish peroxidase; c′, cytochrome c′.1-b In multiheme proteins the positive part of this MCD feature is often obscured by adjacent low spin heme bands; the wavelength of the intensity minimum to longer wavelengths has been quoted. Open table in a new tab Quantitative analysis of the dependence of the 605- and 635-nm bands on the potential at pH 7 to obtain E°′ for each redox species is made difficult by the low intensities and overlapping nature of these bands. In the fully oxidized enzyme (Fig. 4, —) the CT band is clearly positioned at 595 nm. As the dinuclear center starts to become reduced, the 595-nm band begins to decrease in intensity. At +250 mV (Fig. 4, ---) it has decreased to approximately half its original intensity, and a new band has appeared centered at 635 nm. This movement in the CT band represents a change from a μ-oxo bridged species to one where the heme b3 is ligated by His/H2O. The next phase of reduction from +250 mV to +140 mV (Fig. 4, ····) causes the 595-nm band to further decrease in intensity. However, this further decrease does not correspond to a further increase in the 635-nm band but to the appearance of another new band at 605 nm. This represents the formation of a His/OH−-ligated heme b3 from the μ-oxo bridged species. During the remainder of the reduction of the dinuclear center the 605 and 635-nm bands titrate at similar potentials and have completely disappeared by +22 mV with the full reduction of the heme b3 (Fig. 4, ·−·−). This three-phase reduction of the dinuclear center can be clearly seen when fitting the change in absorbance of the 595-nm band against potential (Fig. 5 A). The data can be fitted to three n = 1 Nernst components. The first phase corresponds to the breaking of the μ-oxo bridge, between the FeB and the heme b3 and the associated shift of the 595-nm band to 635 nm, to form the His/H2O-ligated heme b3. This rearrangement of ligation within the dinuclear center has an E°′ of +325 mV. The second phase represents a change in the dinuclear center from the μ-oxo bridged species to one where the heme b3 is His/OH−-ligated and is associated with the shift in the 595-nm band to 605 nm. This phase has an E°′ of +240 mV. The final phase corresponds to the full reduction of both species from a ferric heme b3 to a ferrous heme b3 where the shoulder of the 605 and 635 bands detected at 595 nm disappear. Both processes occur at isopotentials where the E°′ is +50 mV (Fig6). By contrast, at pH 8.5, where only a single species is present (His/OH−) in the three electron-reduced form, a simple two-phase titer is seen (Fig. 5 B). The first phase represents the reduction of the FeB and breaking of the μ-oxo bridge and the second phase represents the full reduction of the high spin His/OH−-ligated heme b3.Figure 5Mediated redox titration of the dinuclear center of NOR. A, titration at pH 7.0. The data fitted to two n = 1 Nernstian components with Emvalues of +299 mV (85%) and +62 mV (15%) is shown by the dashed line. Data fitted to three n = 1 Nernstian components with Em values of +325 mV (53%), +240 mV (32%), and +50 mV (15%) is shown by the solid line. B, titration at pH 8.5 fitted to two n = 1 Nernstian components with Em values of +150 mV (50%) and +20 mV (50%).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Diagram showing a model for the effects of pH and redox state on ligation of the high spin heme b3 of NOR. Midpoint redox potentials correspond to those observed at pH 7.0.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Having identified three spectral forms of the ferric heme b3, it is possible to forward tentative suggestions as to both their origin and their role in possible catalytic cycles of the enzyme. At present there is no agreement on the mechanism of NO reduction by NOR. Arguments have been put forward for the two substrate NO molecules binding to FeB in the so-called cis model (2Hendricks J. Gohlke U. Saraste M. J. Bioenerg. Biomembr. 1998; 30: 15-24Crossref PubMed Scopus (60) Google Scholar). An alternative trans mechanism where one NO molecule binding to each of FeB and heme b3 has also been forwarded (2Hendricks J. Gohlke U. Saraste M. J. Bioenerg. Biomembr. 1998; 30: 15-24Crossref PubMed Scopus (60) Google Scholar). However, in both cases it is likely that at some point in the catalytic cycle a ferric heme b3 bound by a water molecule is formed. The identification of the 635-nm spectral form of heme b3 is the first time such a species has been identified. It can be supposed that this can be derived from the reduction of a μ-oxo bridged Fe(III)-Fe(III) dinuclear center, which would correlate to the 595-nm form of oxidized NOR. It then seems plausible that on reduction of the FeB of the dinuclear center with one electron a proton will also enter the catalytic site leading to a hydroxide-bound ferric heme b3(Fig. 6). MCD studies of this spectral form of the heme b3 are consistent with this assertion. There are many examples of high spin hemes that exhibit CT bands at ∼630 nm, and these characteristically arise from ferric hemes with His-H2O coordination (e.g. in E. coli cytochrome-bo3 oxidase (16Cheng J.C. Osborne G.A. Stephens P.J. Eton W.A. Nature. 1973; 241: 193-194Crossref Scopus (45) Google Scholar)). Thus it seems most likely that the low pH 635-nm form of heme b3arises from a simple protonation of the putative hydroxide-bound high pH 605-nm form (Fig. 6). It is also important to note that experiments in our laboratory have shown that NOR activity in BTP buffer at pH 6.0 is 8-fold higher (∼70 s−1) than at pH 8.5 (∼8 s−1) (data not shown). The acidic nature of this optimal activity is in agreement with previously published data obtained in various buffers (32Heiss B. Frunke K. Zumft W.G. J. Bacteriol. 1998; 171: 3288-3297Crossref Google Scholar, 33Dermashia M. Turk T. Hollocher T.C. J. Biol. Chem. 1991; 266: 10899-10905Abstract Full Text PDF PubMed Google Scholar). At this pH the His-H2O form of the enzyme dominates. The higher midpoint redox potential of this species than that of the His-hydroxide form may allow more rapid electron transfer from physiological electron donors such as cytochrome c and pseudoazurin. Having identified spectral signals that might correspond to distinct intermediates in the catalytic cycle of NOR, it is now necessary to try to trap these spectral forms in rapid-reaction experiments. Such experiments may in turn provide further insight into the N2O-generating half of the catalytic cycle. We thank Ann Reilly, Jeremy Thornton, and David Clark for expert technical contributions and Gareth Butland for helpful discussions. We also thank the EU SENORA groups of Simon de Vries, Matti Saraste, Rob van Spanning, and Costos Varotsis for useful discussions.