Very Early Reaction Intermediates Detected by Microsecond Time Scale Kinetics of Cytochrome cd1-catalyzed Reduction of Nitrite
2008; Elsevier BV; Volume: 283; Issue: 41 Linguagem: Inglês
10.1074/jbc.m804493200
ISSN1083-351X
AutoresKatharine A. Sam, Marc J. F. Strampraad, Simon de Vries, Stuart J. Ferguson,
Tópico(s)Amino Acid Enzymes and Metabolism
ResumoParacoccus pantotrophus cytochrome cd1 is a nitrite reductase found in the periplasm of many denitrifying bacteria. It catalyzes the reduction of nitrite to nitric oxide during the denitrification part of the biological nitrogen cycle. Previous studies of early millisecond intermediates in the nitrite reduction reaction have shown, by comparison with pH 7.0, that at the optimum pH, approximately pH 6, the earliest intermediates were lost in the dead time of the instrument. Access to early time points (∼100 μs) through use of an ultra-rapid mixing device has identified a spectroscopically novel intermediate, assigned as the Michaelis complex, formed from reaction of fully reduced enzyme with nitrite. Spectroscopic observation of the subsequent transformation of this species has provided data that demand reappraisal of the general belief that the two subunits of the enzyme function independently. Paracoccus pantotrophus cytochrome cd1 is a nitrite reductase found in the periplasm of many denitrifying bacteria. It catalyzes the reduction of nitrite to nitric oxide during the denitrification part of the biological nitrogen cycle. Previous studies of early millisecond intermediates in the nitrite reduction reaction have shown, by comparison with pH 7.0, that at the optimum pH, approximately pH 6, the earliest intermediates were lost in the dead time of the instrument. Access to early time points (∼100 μs) through use of an ultra-rapid mixing device has identified a spectroscopically novel intermediate, assigned as the Michaelis complex, formed from reaction of fully reduced enzyme with nitrite. Spectroscopic observation of the subsequent transformation of this species has provided data that demand reappraisal of the general belief that the two subunits of the enzyme function independently. Cytochrome cd1 is a homodimeric enzyme found in the periplasm of denitrifying bacteria such as Paracoccus pantotrophus and Pseudomonas aeruginosa. It catalyzes the one electron reduction of nitrite to nitric oxide, which is the first committed step in the denitrification pathway of the biological nitrogen cycle (1Allen J.W.A. Ferguson S.J. Fülöp V. Messerschmidt A. Huber R. Pulos T. Wieghardt K. Handbook of Metalloproteins. John Wiley and Sons, Ltd., Chichester, UK2001: 424-439Google Scholar, 2Berks B.C. Ferguson S.J. Moir J.W.B. Richardson D.J. Biochim. Biophys. Acta. 1995; 1232: 97-173Crossref PubMed Scopus (495) Google Scholar). There are two classes of enzyme that catalyze this reaction. The copper nitrite reductases, comprising the first category, contain an electron-accepting type I copper center and a type II copper center at the active site (3Kukimoto M. Nishiyama M. Murphy M.E. Turley S. Adman E.T. Horinouchi S. Beppu T. Biochemistry. 1994; 33: 5246-5252Crossref PubMed Scopus (150) Google Scholar). Cytochromes cd1 belong to a second group of nitrite reductase (1Allen J.W.A. Ferguson S.J. Fülöp V. Messerschmidt A. Huber R. Pulos T. Wieghardt K. Handbook of Metalloproteins. John Wiley and Sons, Ltd., Chichester, UK2001: 424-439Google Scholar, 2Berks B.C. Ferguson S.J. Moir J.W.B. Richardson D.J. Biochim. Biophys. Acta. 1995; 1232: 97-173Crossref PubMed Scopus (495) Google Scholar). These are heme-containing enzymes with one c heme and one d1 heme per monomer. The c heme is the site of electron donation from external electron donor proteins; for the enzyme from P. pantotrophus, these have been shown to be pseudoazurin and cytochrome c550 (4Pearson I.V. Page M.D. van Spanning R.J. Ferguson S.J. J. Bacteriol. 2003; 185: 6308-6315Crossref PubMed Scopus (47) Google Scholar). The d1 heme forms the active site of the enzyme. The 1.55-Å crystal structure of oxidized cytochrome cd1 from P. pantotrophus reveals that in its oxidized as-isolated state, the c heme is axially ligated by histidines 69 and 17, and the d1 heme binds histidine 200 and tyrosine 25 (5Fülöp V. Moir J.W.B. Ferguson S.J. Hajdu J. Cell. 1995; 81: 369-377Abstract Full Text PDF PubMed Scopus (251) Google Scholar). The c heme is located in a predominantly α-helical domain of the enzyme, whereas the d1 heme resides in a β-propeller structure. The tyrosine ligand to the d1 heme is part of the N-terminal c heme domain and is connected to the c heme distal ligand, His-17, by a short polypeptide loop of just 8 amino acids. Upon reduction of the enzyme, His-17 is replaced by Met-106, and Tyr-25 dissociates leaving the active site pentacoordinate and able to bind substrate (5Fülöp V. Moir J.W.B. Ferguson S.J. Hajdu J. Cell. 1995; 81: 369-377Abstract Full Text PDF PubMed Scopus (251) Google Scholar).Our previous study of nitrite reduction by P. pantotrophus cytochrome cd1 described the use of stopped flow methodology to study the kinetics of nitrite reduction by P. pantotrophus cytochrome cd1 (6Sam K.A. Tolland J.D. Fairhurst S.A. Higham C.W. Lowe D.J. Thorneley R.N.F. Allen J.W.A. Ferguson S.J. Biochem. Biophys. Res. Commun. 2008; 371: 719-723Crossref PubMed Scopus (8) Google Scholar). At the earliest time point (2–3 ms) using a conventional stopped flow apparatus, at pH 6.0, a significant proportion of the enzyme had already undergone one turnover. At this time point three separate species were assigned at the d1 heme, Fe(II)-NO, Fe(II)-NO+, and ferrous d1 heme, with neither substrate nor NO bound (6Sam K.A. Tolland J.D. Fairhurst S.A. Higham C.W. Lowe D.J. Thorneley R.N.F. Allen J.W.A. Ferguson S.J. Biochem. Biophys. Res. Commun. 2008; 371: 719-723Crossref PubMed Scopus (8) Google Scholar). The latter, it was postulated, was an intermediate of the enzyme that had released product generated in the first turnover. The findings of this conventional stopped flow study indicated that a faster technique was required to observe intermediates of the first turnover.A rapid freezing technique known as microsecond freeze-hyperquenching (MHQ) 2The abbreviations used are: MHQ, microsecond freeze-hyperquenching; FTIR, Fourier-transform infrared. 2The abbreviations used are: MHQ, microsecond freeze-hyperquenching; FTIR, Fourier-transform infrared. has been developed in the de Vries laboratory (7Cherepanov A.V. De Vries S. Biochim. Biophys. Acta. 2004; 1656: 1-31Crossref PubMed Scopus (74) Google Scholar, 8Wiertz F.G. Richter O.M. Ludwig B. de Vries S. J. Biol. Chem. 2007; 282: 31580-31591Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). This technique allows mixing and freezing of enzyme and substrate on a microsecond time scale, with an effective dead time of 75 μs (8Wiertz F.G. Richter O.M. Ludwig B. de Vries S. J. Biol. Chem. 2007; 282: 31580-31591Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), and thus enables previously undetectable intermediates to be observed. We report a completely novel species observed at the d1 heme just 130 μs after mixing of the fully reduced enzyme with nitrite in the absence of excess reductant. The formation of other early intermediates was also observed within the first 800 μs of the reaction, and novel insight into their involvement in the catalytic cycle is presented; these intermediates provide evidence that the long standing assumption that the two monomers are independent needs reappraisal.EXPERIMENTAL PROCEDURESProduction of Cytochrome cd1—P. pantotrophus was grown under anaerobic conditions at 37 °C. Cytochrome cd1 was purified from the periplasm of the cells according to the method of Moir et al. (9Moir J.W.B. Baratta D. Richardson D.J. Ferguson S.J. Eur. J. Biochem. 1993; 212: 377-385Crossref PubMed Scopus (118) Google Scholar) as modified by Koppenhöfer et al. (10Koppenhöfer A. Little R.H. Lowe D.J. Ferguson S.J. Watmough N.J. Biochemistry. 2000; 39: 4028-4036Crossref PubMed Scopus (35) Google Scholar). The purity of the enzyme was determined by the Rz value (A406/A280), and all cytochrome cd1 used in this work had an Rz of >1.25. The concentration of the enzyme was determined at 406 nm for the oxidized enzyme and 418 nm for the reduced, with the respective extinction coefficients of 142.5 mm–1 (11Kobayashi K. Koppenhöfer A. Ferguson S.J. Tagawa S. Biochemistry. 1997; 36: 13611-13616Crossref PubMed Scopus (60) Google Scholar) and 161.5 mm–1 (10Koppenhöfer A. Little R.H. Lowe D.J. Ferguson S.J. Watmough N.J. Biochemistry. 2000; 39: 4028-4036Crossref PubMed Scopus (35) Google Scholar). These extinction coefficients refer to the concentration of the enzyme monomer. Throughout this work the monomeric concentration will be reported.Anaerobic Preparation of the Enzyme for Rapid Kinetic Experiments—The purified enzyme was transferred to an anaerobic glove box (Coy Laboratory Products Inc.) that was maintained at less than 2 ppm O2. Cytochrome cd1 was reduced with a small excess of sodium dithionite and then passed down a desalting column packed with P6-DG resin (Bio-Rad) and equilibrated with 50 mm potassium phosphate of the desired pH. All buffers were sparged overnight in the anaerobic glove box to remove oxygen. The enzyme was loaded into a gas-tight syringe that had been presoaked in sodium dithionite and washed to remove traces of excess reductant. The absence of excess dithionite was confirmed by testing buffer expelled from the syringe with methyl viologen, which turns blue on contact with dithionite. 10 mm potassium nitrite was made up in 50 mm potassium phosphate buffer of the desired pH and loaded into a second gas-tight syringe that had undergone the same process as the enzyme-containing syringe to remove traces of oxygen. This was important because cytochrome cd1 also functions as an oxidase (10Koppenhöfer A. Little R.H. Lowe D.J. Ferguson S.J. Watmough N.J. Biochemistry. 2000; 39: 4028-4036Crossref PubMed Scopus (35) Google Scholar).MHQ—MHQ measurements were performed as described previously (7Cherepanov A.V. De Vries S. Biochim. Biophys. Acta. 2004; 1656: 1-31Crossref PubMed Scopus (74) Google Scholar) and as modified by Ref. 24Kharitonov V.G. Sharma V.S. Magde D. Koesling D. Biochemistry. 1997; 36: 6814-6818Crossref PubMed Scopus (163) Google Scholar. Optical measurements were performed on an SLM-Aminco DW2000 scanning spectrophotometer, which was adapted for low temperature measurements of spectra of the frozen powders obtained from the MHQ experiments; the entire MHQ design and set up of the spectrophotometer are described in Ref. 7Cherepanov A.V. De Vries S. Biochim. Biophys. Acta. 2004; 1656: 1-31Crossref PubMed Scopus (74) Google Scholar.RESULTSFully reduced cytochrome cd1 was mixed with potassium nitrite in the absence of excess reductant in the MHQ apparatus. Because cytochrome cd1 also functions as an oxidase (10Koppenhöfer A. Little R.H. Lowe D.J. Ferguson S.J. Watmough N.J. Biochemistry. 2000; 39: 4028-4036Crossref PubMed Scopus (35) Google Scholar), great care was taken to ensure the equipment was sufficiently anaerobic to prevent re-oxidation of the enzyme, from which excess reductant had been removed, before mixing. To test that this requirement had been met, the enzyme was mixed with anaerobic phosphate buffer in the absence of excess reductant, and the optical spectrum produced was compared with the spectrum recorded when the enzyme was mixed with phosphate buffer in the presence of excess reductant. The position of the c heme Soret band (418 nm), which is characteristic of reduced c heme in this enzyme, and the d1 heme spectral features at 460 and 653 nm both indicated that the enzyme remained fully reduced in the apparatus, even in the absence of excess reductant (Fig. 1 and Fig. 3 (t = 0 spectra)).FIGURE 3Optical spectra measured at the times indicated after mixing of fully reduced P. pantotrophus cytochrome cd1 (in the absence of excess reductant) and 10 mm potassium nitrite (which was in at least 10-fold excess over enzyme monomer) at pH 6.0. The dashed lines indicate 653 nm, the position of the d1 heme peak in the fully reduced spectrum. The dotted lines indicate 620 nm. The region between 500 and 700 nm is multiplied by 2.5. Mixing was carried out at 25 °C. Spectra were measured at 77 K.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fully reduced cytochrome cd1 was mixed with potassium nitrite in the MHQ apparatus at pH 7.0, and the reaction was quenched at various time points between 130 μs and 11 ms after mixing to build up a profile of the early reaction intermediates (Fig. 1). A spectrum taken at the fastest possible time point (∼75 μs) was not different from the 130-μs spectrum, and therefore we have concentrated our analysis on time points ≥130 μs, which can be reported with the greatest accuracy (±5%). 130 μs after mixing of enzyme and substrate, a peak at 620 nm was observed. This peak is shifted by 33 nm from the corresponding d1 heme absorbance at 653 nm in the starting spectrum of the fully reduced enzyme (Fig. 1). The d1 heme peak at 460 nm became a shoulder. Complexes of NO and CO bound to the ferrous enzyme from P. aeruginosa lose absorbance at 460 nm altogether (12Das T.K. Wilson E.K. Cutruzzolà F. Brunori M. Rousseau D.L. Biochemistry. 2001; 40: 10774-10781Crossref PubMed Scopus (26) Google Scholar), and the spectrum of the complex of CN– bound to ferrous P. pantotrophus cytochrome cd1 is also significantly perturbed in this region (13Jafferji A. Allen J.W.A. Ferguson S.J. Fülöp V. J. Biol. Chem. 2000; 275: 25089-25094Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). We therefore interpret the loss of absorbance at 460 nm as ligand binding at the active site. At this time point, the c heme was still fully reduced, is judged by the position of the Soret band. The kcat of cytochrome cd1 is 72 s–1 per enzyme monomer at pH 7.0 (14Richter C.D. Allen J.W.A. Higham C.W. Koppenhöfer A. Zajicek R.S. Watmough N.J. Ferguson S.J. J. Biol. Chem. 2002; 277: 3093-3100Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), which means that the catalytic cycle takes place over a period of about 14 ms. 130 μs was therefore extremely early in the catalytic cycle of cytochrome cd1-catalyzed nitrite reduction. An absorbance maximum at around 620 nm has not been observed during conventional studies of the kinetics or ligand binding for this enzyme, and thus it is concluded that the species observed 130 μs after mixing of enzyme and substrate was very probably the Michaelis complex of fully reduced cytochrome cd1 with nitrite bound at the d1 heme. This assignment is supported by further spectroscopic evidence that will be discussed later. At pH 7.0, between 130 and 780 μs, the 620 nm peak began to red shift but was still predominant in the spectrum until 780 μs. By 1.31 ms, two separate peaks of roughly equal intensity were visible at 630 and 660 nm. This spectrum is comparable with that seen in the earliest time point, ∼3 ms, observed in conventional stopped flow studies (6Sam K.A. Tolland J.D. Fairhurst S.A. Higham C.W. Lowe D.J. Thorneley R.N.F. Allen J.W.A. Ferguson S.J. Biochem. Biophys. Res. Commun. 2008; 371: 719-723Crossref PubMed Scopus (8) Google Scholar, 15George S.J. Allen J.W.A. Ferguson S.J. Thorneley R.N.F. J. Biol. Chem. 2000; 275: 33231-33237Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). However, presumably because the current spectra were recorded at low temperature, the two separate peaks observed at this time point are much more clearly resolved than in the room temperature spectrum. The 630-nm peak can be assigned with confidence as arising from d1 Fe(II)-NO+ because its formation has previously been observed to occur with the same rate constant as the species shown to be Fe(II)-NO+ by FTIR; note that Fe(II)-NO+, while isoelectronic with Fe(III)-NO, is a distinct species, and the previously reported FTIR band observed at 1913 cm–1 is assigned to the former (15George S.J. Allen J.W.A. Ferguson S.J. Thorneley R.N.F. J. Biol. Chem. 2000; 275: 33231-33237Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). It has not proved possible to assign unequivocally the 660 nm species to date. However, the best current interpretation is that it arises from a high spin ferrous d1 heme that is pentacoordinate or more probably has a 6th weak field ligand such as water (6Sam K.A. Tolland J.D. Fairhurst S.A. Higham C.W. Lowe D.J. Thorneley R.N.F. Allen J.W.A. Ferguson S.J. Biochem. Biophys. Res. Commun. 2008; 371: 719-723Crossref PubMed Scopus (8) Google Scholar).The c heme remained essentially reduced for the first 410 μs of the reaction and then underwent oxidation between 410 μs and 1.31 ms. It then continued to oxidize further but at a much slower rate until the final time point at 11 ms. This second slower phase of oxidation was interpreted as a balance of continued oxidation in conjunction with the back reduction of the c heme shown by George et al. (15George S.J. Allen J.W.A. Ferguson S.J. Thorneley R.N.F. J. Biol. Chem. 2000; 275: 33231-33237Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). After 11 ms, the c heme had reached ∼55% oxidation (Fig. 1), which is again in good agreement with the conventional stopped flow studies of the nitrite reduction reaction at this pH (6Sam K.A. Tolland J.D. Fairhurst S.A. Higham C.W. Lowe D.J. Thorneley R.N.F. Allen J.W.A. Ferguson S.J. Biochem. Biophys. Res. Commun. 2008; 371: 719-723Crossref PubMed Scopus (8) Google Scholar, 15George S.J. Allen J.W.A. Ferguson S.J. Thorneley R.N.F. J. Biol. Chem. 2000; 275: 33231-33237Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar).Conventionally, the percentage oxidation of P. pantotrophus cytochrome cd1 is judged by the relative intensity of the c heme α-band. However, this requires that spectra are first normalized according to protein concentration. Using the MHQ technique, it was not possible to determine the concentration of the enzyme in the final sample from which the optical spectra were produced because an unknown and slightly variable amount of cold reacted powder was mixed with cold isopentane. The percentage oxidation of the c heme of the enzyme can therefore only be judged by the position of the Soret band. The Soret band of P. pantotrophus cytochrome cd1 shifts from 410 to 418 nm between its fully oxidized (His/Met coordinated) and fully reduced forms. Assuming a linear relationship between oxidation state and Soret position, the extent of c heme oxidation was estimated from the percentage Soret shift; however, the values obtained are inevitably approximate.The final species seen after 11 ms at pH 7.0 still contains clear contributions from the 630 and 660 nm species that are also observed in the conventional time stopped flow experiments at this time point. It has been shown previously from later time points (6Sam K.A. Tolland J.D. Fairhurst S.A. Higham C.W. Lowe D.J. Thorneley R.N.F. Allen J.W.A. Ferguson S.J. Biochem. Biophys. Res. Commun. 2008; 371: 719-723Crossref PubMed Scopus (8) Google Scholar, 15George S.J. Allen J.W.A. Ferguson S.J. Thorneley R.N.F. J. Biol. Chem. 2000; 275: 33231-33237Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) that this species decays over a period of 100 ms to produce a spectrum containing a single peak in the d1 heme region with maximum absorbance at 630 nm. Stopped flow FTIR shows that the peak corresponding to d1 Fe(II)-NO+ forms at the same rate as the optical species at 630 nm, and therefore, as previously explained, the 630 nm absorbing species is assigned as containing d1 Fe(II)-NO+ (15George S.J. Allen J.W.A. Ferguson S.J. Thorneley R.N.F. J. Biol. Chem. 2000; 275: 33231-33237Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar).The mixing of fully reduced cytochrome cd1 (in the absence of excess reductant) with potassium nitrite was repeated at pH 6.0 because our recent study of the pH dependence of the nitrite reduction reaction on a slower time scale showed that at pH 6.0 the final reaction product was different and formed approximately three times faster compared with pH 7.0. The data in the current ultra-rapid reaction study show that at pH 6.0 a similar pattern of early intermediates to those at pH 7.0 was observed (Fig. 3). However, their lifetimes differed significantly from those at pH 7.0. As with the reaction at pH 7.0, a 620-nm peak was observed in the optical spectrum of the fastest time point (130 μs), which as before is assigned as the Michaelis complex of c Fe(II)·d1Fe(II)-NO–2. At pH 6.0, oxidation of the c heme was similar to pH 7.0 with the majority of this oxidation occurring between 780 μs and 1.31 ms (Fig. 2). The most notable difference between pH 6.0 and 7.0 is that at pH 6.0 the species with a peak at 660 nm decayed on a much faster time scale (compare Fig. 1 and Fig. 3). At both pH 6.0 and pH 7.0, a small amount of absorbance at 660 nm was evident in the spectrum at 780 μs. By 1.31 ms in the pH 6.0 reaction, which is the time point at which the greatest level of c heme oxidation was observed, clear resolution of a peak at 630 nm, which was assigned as d1 Fe(II)-NO+, and an increase in intensity of the 660 nm peak occurred. Unlike at pH 7.0, where both the 660 and 630 nm absorbance peaks remained for longer than 11 ms after mixing of enzyme and substrate, at pH 6.0, the 660 nm peak decayed much faster and had substantially disappeared by 2.2 ms, leaving a species generating a single 630 nm peak by 11 ms.FIGURE 2Percentage oxidation of the c heme at pH 6.0 (—) and pH 7.0 (- - -) at specific time points (indicated in Figs. 1 and 3) during the reaction of P. pantotrophus cytochrome cd1 with potassium nitrite, which was in ∼10-fold excess to enzyme monomer. Enzyme and substrate were mixed in the MHQ apparatus at 25 °C, and the reaction was quenched at various time points. The percentage oxidation of the c heme was measured as the % shift between 418 nm (fully reduced His/Met coordinated) and 410 nm (fully oxidized His/Met coordinated).View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONThis work exemplifies the novel insight into enzyme mechanisms, in this case cytochrome cd1, which is available through the use of the recently developed MHQ apparatus. The novel species observed just 130 μs after mixing of enzyme and substrate, with an optical d1 heme signature at 620 nm, is argued to be the Michaelis complex of fully reduced enzyme with nitrite bound at the d1 heme, cFe(II)⋅d1Fe(II)−NO2−. A peak at 620 nm has not previously been observed during the catalytic cycle of cytochrome cd1, and indeed there are very few references to d1 heme complexes with absorbance maxima below 630 nm. The closest reported d1 heme absorbance maximum to 620 nm is that of the d1 heme pyridine hemochrome, d1Fe(II)-bis-pyr, the absorbance maximum of which has been reported at 617 nm (16Newton N. Biochem. J. 1967; 105: C21-C23Crossref Scopus (13) Google Scholar) and 620 nm (17Yamanaka T. Okunuki K. Biochim. Biophys. Acta. 1963; 67: 407-416Crossref PubMed Google Scholar). Also of note is that ferrous, cyanide-bound cytochrome cd1 has an absorbance maximum at 628 nm (13Jafferji A. Allen J.W.A. Ferguson S.J. Fülöp V. J. Biol. Chem. 2000; 275: 25089-25094Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 18Allen J.W. Higham C.W. Zajicek R.S. Watmough N.J. Ferguson S.J. Biochem. J. 2002; 366: 883-888Crossref PubMed Google Scholar). Both the pyridine hemochrome and the ferrous CN–-bound species are examples of ferrous d1 heme with strong field ligands. Nitrite is a strong field π -acceptor ligand, and the fact that these two complexes of ferrous d1 heme, with strong field axial ligands, result in observed absorbance maxima below 630 nm supports the proposal that the 620-nm peak seen in this study arises from nitrite bound to ferrous d1 heme, the Michaelis complex. The time scale on which the 620-nm peak is present (between 130 and 780 μs after mixing of enzyme and substrate) is also consistent with it being the Michaelis complex, given the length of the catalytic cycle, which is in the order of 14 ms at pH 7.0 and 8 ms at pH 6.0, as determined by Richter et al. (14Richter C.D. Allen J.W.A. Higham C.W. Koppenhöfer A. Zajicek R.S. Watmough N.J. Ferguson S.J. J. Biol. Chem. 2002; 277: 3093-3100Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). This also makes it unlikely that the first molecule of product NO is formed within the first 130 μs. The c heme apparently remains fully reduced at the first time point of 130 μs; thus no intramolecular electron transfer has occurred by this stage. Therefore, the only possibilities, other than the Michaelis complex, for the species absorbing at 620 nm, are ferric d1 heme with either NO or nitrite bound; however, the above discussion of kinetic parameters argues against these possibilities. As reported in previous work (19Sam K.A. Fairhurst S.A. Thorneley R.N.F. Allen J.W.A. Ferguson S.J. J. Biol. Chem. 2008; 283: 12555-12563Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), model spectra were obtained for the oxidized Y25S cytochrome cd1 with nitrite and NO bound; it was not possible to use the wild type enzyme for these experiments because tyrosine 25 blocks the active site preventing exogenous ligand coordination to the oxidized enzyme. The Y25S enzyme is competent to exogenous ligand binding in its oxidized as-isolated state, and therefore circumvents this problem. No peak between 550 and 630 nm was seen for the Y25S enzyme with either nitrite or NO bound, further indicating that the 620 nm peak does not arise from either of these ligands bound to ferric d1 heme. Recent work has invoked the possibility of electron donation from an amino acid side chain during nitrite reduction by cytochrome cd1 (20van Wonderen J.H. Knight C. Oganesyan V.S. George S.J. Zumft W.G. Cheesman M.R. J. Biol. Chem. 2007; 282: 28207-28215Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar), and therefore formation of cFe(II)·d1Fe(II)-NO was also addressed. However, when the 130-μs sample was subjected to EPR, no signal arising from Fe(II)-NO was observed. The only logical assignment of the 620 nm peak, therefore, remains the Michaelis complex.At pH 7.0, following observation of the proposed Michaelis complex at 130 μs, the 620 nm absorbance maximum is seen to red shift and flatten. This is concurrent with another species being formed at the d1 heme, the absorbance of which is not sufficiently separated from 620 nm to distinguish formation of a separate peak. There is considerable evidence for non-equivalence between monomers of P. pantotrophus cytochrome cd1 (13Jafferji A. Allen J.W.A. Ferguson S.J. Fülöp V. J. Biol. Chem. 2000; 275: 25089-25094Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 21Sjögren T. Hajdu J. J. Biol. Chem. 2001; 276: 13072-13076Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 22Williams P.A. Fülöp V. Garman E.F. Saunders N.F. Ferguson S.J. Hajdu J. Nature. 1997; 389: 406-412Crossref PubMed Scopus (259) Google Scholar), but the general assumption has been that the monomers are kinetically independent. However, it is possible that the residual absorbance at 620 nm, in conjunction with increased absorbance at longer wavelength, reflects nitrite reduction occurring at monomer 1, whereas the Michaelis complex remains at monomer 2. By 1.31 ms, significant absorbance has become apparent at 660 nm, and the absorbance between 620 and 630 nm remains broad and flat although it is further shifted toward 630 nm. By 2.2 ms, the previously apparent absorbance at 620 nm appears to have significantly diminished, with the emergence of a much sharper peak at 630 nm and further increase in the absorbance at 660 nm. In previous work we have tentatively assigned the species with 660 nm absorbance as a high spin ferrous d1 heme, which is either pentacoordinate or which has a 6th weak field ligand such as a water (6Sam K.A. Tolland J.D. Fairhurst S.A. Higham C.W. Lowe D.J. Thorneley R.N.F. Allen J.W.A. Ferguson S.J. Biochem. Biophys. Res. Commun. 2008; 371: 719-723Crossref PubMed Scopus (8) Google Scholar). The reason for the uncertainty of this assignment was because it is hard to justify how the d1 heme, in the presence of a large excess of nitrite, could have anything bound other than this anion. However, in light of these new insights into much earlier time points in the reaction, several steps in the nitrite reduction reaction may, in fact, be gated by events occurring at the other cytochrome cd1 monomer. The evidence for this is as follows: as already discussed, the earliest time point is assigned as the Michaelis complex of fully reduced enzyme with nitrite bound at the d1 heme. Later time points show that this absorbance shifts to longer wavelengths, but no clear decrease in absorbance at 620 nm is observed until formation of the 660 nm species, at which time, clear absorbance at 630 nm is observed with a marked decrease in absorbance at 620 nm. A peak at 630 nm, later on in the reaction between cytochrome cd1 and nitrite, has been shown to correspond to ferric d1 heme with NO bound, formally d1 Fe(II)-NO+. An interpretation of the observed absorbance changes at the d1 heme is that reduction of nitrite to NO at monomer 2 cannot occur until product has dissociated from monomer 1. Hence, residual absorbance at 620 nm, arising from the Michaelis complex at monomer 2, is observed until product dissociation occurs at monomer 1. If the assignment of the 660 nm species as ferrous pentacoordinate d1 heme is accurate, then the product dissociated state of monomer 1 is evident from the 6
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