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• W2074037790 abstract "The single turnover of (1R)(+)-camphor-bound oxyferrous cytochrome P450-CAM with one equivalent of dithionite-reduced putidaredoxin (Pdx) was monitored for the appearance of transient intermediates at 3 °C by double mixing rapid scanning stopped-flow spectroscopy. With excess camphor, three successive species were observed after generating oxyferrous P450-CAM and reacting versus reduced Pdx: a perturbed oxyferrous derivative, a species that was a mixture of high and low spin Fe(III), and high spin ferric camphor-bound enzyme. The rates of the first two steps, ∼140 and ∼85 s-1, were assigned to formation of the perturbed oxyferrous intermediate and to electron transfer from reduced Pdx, respectively. In the presence of stoichiometric substrate, three phases with similar rates were seen even though the final state is low spin ferric P450-CAM. This is consistent with substrate being hydroxylated during the reaction. The single turnover reaction initiated by adding dioxygen to a preformed reduced P450-CAM·Pdx complex with excess camphor also led to phases with similar rates. It is proposed that formation of the perturbed oxyferrous intermediate reflects alteration of H-bonding to the proximal Cys, increasing the reduction potential of the oxyferrous state and triggering electron transfer from reduced Pdx. This species may be a direct spectral signature of the effector role of Pdx on P450-CAM reactivity (i.e. during catalysis). The substrate-free oxyferrous enzyme also reacted readily with reduced Pdx, showing that the inability of substrate-free P450-CAM to accept electrons from reduced Pdx and function as an NADH oxidase is completely due to the incapacity of reduced Pdx to deliver the first but not the second electron. The single turnover of (1R)(+)-camphor-bound oxyferrous cytochrome P450-CAM with one equivalent of dithionite-reduced putidaredoxin (Pdx) was monitored for the appearance of transient intermediates at 3 °C by double mixing rapid scanning stopped-flow spectroscopy. With excess camphor, three successive species were observed after generating oxyferrous P450-CAM and reacting versus reduced Pdx: a perturbed oxyferrous derivative, a species that was a mixture of high and low spin Fe(III), and high spin ferric camphor-bound enzyme. The rates of the first two steps, ∼140 and ∼85 s-1, were assigned to formation of the perturbed oxyferrous intermediate and to electron transfer from reduced Pdx, respectively. In the presence of stoichiometric substrate, three phases with similar rates were seen even though the final state is low spin ferric P450-CAM. This is consistent with substrate being hydroxylated during the reaction. The single turnover reaction initiated by adding dioxygen to a preformed reduced P450-CAM·Pdx complex with excess camphor also led to phases with similar rates. It is proposed that formation of the perturbed oxyferrous intermediate reflects alteration of H-bonding to the proximal Cys, increasing the reduction potential of the oxyferrous state and triggering electron transfer from reduced Pdx. This species may be a direct spectral signature of the effector role of Pdx on P450-CAM reactivity (i.e. during catalysis). The substrate-free oxyferrous enzyme also reacted readily with reduced Pdx, showing that the inability of substrate-free P450-CAM to accept electrons from reduced Pdx and function as an NADH oxidase is completely due to the incapacity of reduced Pdx to deliver the first but not the second electron. The cytochrome P-450 family of heme-containing mono-oxygenases is involved in the metabolism of xenobiotics and in the production of physiologically important molecules (1Sono M. Roach M.P. Coulter E.D. Dawson J.H. Chem. Rev. 1996; 96: 2841-2887Crossref PubMed Scopus (2091) Google Scholar). A defining characteristic of the P-450 family is the proximal thiolate-ligated heme. P450-CAM (P450-CAM, CYP101) 3The abbreviations used are: P450-CAMcytochrome P450 CYP101 isolated from Pseudomonas putidaPdxputidaredoxinMOPS4-morpholinepropanesulfonic acid.3The abbreviations used are: P450-CAMcytochrome P450 CYP101 isolated from Pseudomonas putidaPdxputidaredoxinMOPS4-morpholinepropanesulfonic acid. from Pseudomonas putida catalyzes the hydroxylation of (1R)(+)-camphor to form (1R)(+)-5-exo-hydroxycamphor (Reaction 1). The electrons required for the reaction flow from NADH to the flavoprotein, putidaredoxin reductase, then to the iron-sulfur (2Fe/2S) protein, putidaredoxin (Pdx), and finally to P450-CAM. In the widely quoted P450 reaction cycle shown in Fig. 1 (1Sono M. Roach M.P. Coulter E.D. Dawson J.H. Chem. Rev. 1996; 96: 2841-2887Crossref PubMed Scopus (2091) Google Scholar), substrate binding converts the ferric low spin resting state (1) to the ferric high spin form (2). Electron transfer from reduced Pdx gives high spin deoxyferrous P-450 (3). Dioxygen binding yields the oxy-ferrous adduct, a ferrous-O2/ferric superoxide resonance hybrid (4a ↔ 4b). CO binding to 3 generates the ferrous-CO derivative (5). Addition of the second electron from reduced Pdx has been proposed to yield a ferric peroxo species (6a), protonation of which gives the hydroperoxo state (6b). Protonation of the distal oxygen produces water and compound I, a ferryl porphyrin π-cation radical (7). This highly oxidizing intermediate abstracts a hydrogen atom from the substrate onto the ferryl oxygen to produce a substrate radical. Radical recombination yields the oxygenated product, and the addition of H2O reforms 1.FIGURE 1Reaction cycle of cytochrome P-450 including postulated intermediates. The porphyrin macrocycle is abbreviated as a parallelogram of nitrogens. States 1, 2, and 7 are neutral (the dot and positive charge on 7 indicate the porphyrin π-cation radical). The overall charge on states 3, 4a, 4b, 5, and 6b is -1, and that on 6a is -2. Paths A and B represent “short circuit” routes to product formation, and paths C and D are uncoupling pathways. Also included are reduction potentials, E½, for P450-CAM and putidaredoxin taken from Ref. 1Sono M. Roach M.P. Coulter E.D. Dawson J.H. Chem. Rev. 1996; 96: 2841-2887Crossref PubMed Scopus (2091) Google Scholar.View Large Image Figure ViewerDownload Hi-res image Download (PPT) cytochrome P450 CYP101 isolated from Pseudomonas putida putidaredoxin 4-morpholinepropanesulfonic acid. cytochrome P450 CYP101 isolated from Pseudomonas putida putidaredoxin 4-morpholinepropanesulfonic acid. Intermediates 6 and 7 are apparently too reactive to build up during turnover, and thus, have never been seen during catalysis. Nonetheless, 6a and 6b have been detected by Davydov et al. (2Davydov R. Macdonald I.D. Makris T.M. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 1999; 121: 10654-10655Crossref Scopus (137) Google Scholar, 3Davydov R. Makris T.M. Kofman V. Werst D.E. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 2001; 123: 1403-1415Crossref PubMed Scopus (405) Google Scholar) in low temperature EPR experiments in which oxyferrous P450-CAM (4b) is reduced by hydrated electrons generated by γ-radiation. Egawa et al. (4Egawa T. Shimada H. Ishimura Y. Biochem. Biophys. Res. Commun. 1994; 201: 1464-1468Crossref PubMed Scopus (179) Google Scholar) have detected P-450 compound I (7) by reacting substrate-free ferric P450-CAM (1) with m-chloroperoxybenzoic acid to yield a species that is spectrally similar to the compound I of another thiolate-ligated heme enzyme, Caldariomyces fumago chloroperoxidase (5Palcic M.M. Rutter R. Araiso T. Hager L.P. Dunford H.B. Biochem. Biophys. Res. Commun. 1980; 94: 1123-1127Crossref PubMed Scopus (114) Google Scholar). Sligar and co-workers (6Kellner D.G. Hung S.-C. Weiss K.E. Sligar S.G. J. Biol. Chem. 2002; 277: 9641-9644Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) have reported similar results with thermostable Cyp119. Shünemann et al. (7Shünemann V. Jung C. Trautwein A.X. Mandon D. Weiss R. FEBS Lett. 2000; 479: 149-154Crossref PubMed Scopus (65) Google Scholar, 8Shünemann V. Jung C. Terner J. Trautwein A.X. Weiss R. J. Inorg. Biochem. 2002; 91: 586-596Crossref PubMed Scopus (61) Google Scholar) have observed a ferryl heme species with substrate-free P450-CAM in reactions with peroxyacetic acid using rapid freeze quenching techniques, but instead of a classical P450-CAM compound I, they reported formation of compound II plus a tyrosine radical, analogous to the compound ES species observed when cytochrome c peroxidase reacts with hydrogen peroxide (1Sono M. Roach M.P. Coulter E.D. Dawson J.H. Chem. Rev. 1996; 96: 2841-2887Crossref PubMed Scopus (2091) Google Scholar). Spolitak et al. (9Spolitak T. Dawson J.H. Ballou D.P. J. Biol. Chem. 2005; 280: 20300-20309Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) have shown that this compound ES-like species evolves from compound I in an acid-dependent step, presumably by abstracting an electron (or hydrogen atom) from a nearby tyrosine residue. The cryogenic crystal structure (10Schlichting I. Berendzen J. Chu K. Stock A.M. Maves S.A. Benson D.E. Sweet B.M. Ringe D. Petsko G.A. Sligar S.G. Science. 2000; 287: 1615-1622Crossref PubMed Scopus (1204) Google Scholar) of oxyferrous P450-CAM exposed to x-rays under conditions known to produce hydrated electrons may possibly be that of P-450 compound I (7), although other species such as 1 cannot be entirely ruled out. Pdx not only delivers electrons to P450-CAM, it is also an effector of catalysis (11Lipscomb J.D. Sligar S.G. Namtvedt M.J. Gunsalus I.C. J. Biol. Chem. 1976; 251: 1116-1124Abstract Full Text PDF PubMed Google Scholar, 12Lipscomb J.D. Biochemistry. 1980; 19: 3590-3599Crossref PubMed Scopus (159) Google Scholar, 13Shimada H. Nagano S. Hori H. Ishimura Y. J. Inorg. Biochem. 2001; 83: 255-260Crossref PubMed Scopus (34) Google Scholar, 14Brazeau B.J. Wallar B.J. Lipscomb J.D. Biochem. Biophys. Res. Commun. 2003; 312: 143-148Crossref PubMed Scopus (31) Google Scholar). In this regard, Pochapsky et al. (15Pochapsky S.S. Pochapsky T.C. Wei J.W. Biochemistry. 2003; 42: 5649-5656Crossref PubMed Scopus (76) Google Scholar) have proposed that Pdx helps prevent uncoupling of electron transfer from product formation. It has been shown that in catalysis, putidaredoxin reductase does not form a ternary complex (putidaredoxin reductase/Pdx/P450-CAM) (16Purdy M.M. Koo L.S. Ortiz de Montellano P.R. Klinman J.P. Biochemistry. 2004; 43: 271-281Crossref PubMed Scopus (37) Google Scholar). Therefore, single turnover experiments to probe the oxygenation mechanism using Pdx and P450-CAM in the absence of putidaredoxin reductase are likely to be relevant to the natural catalytic events. In resonance Raman (17Unno M. Christian J.F. Benson D.E. Gerber N.C. Sligar S.G. Champion P.M. J. Am. Chem. Soc. 1997; 119: 6614-6620Crossref Scopus (76) Google Scholar, 18Unno M. Christian J.F. Sjodin T. Benson D.E. Macdonald I.D.G Sligar S.G. Champion P.M. J. Biol. Chem. 2002; 277: 2547-2553Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), IR (19Nagano S. Shimada H. Tarumi A. Hishiki T. Kimata-Ariga Y. Egawa T. Suematsu M. Park S.Y. Adachi S. Shiro Y. Ishimura Y. Biochemistry. 2003; 42: 14507-14514Crossref PubMed Scopus (50) Google Scholar), NMR (20Tosha T. Yoshioka S. Takahashi S. Ishimori K. Shimada H. Morishima I. J. Biol. Chem. 2003; 278: 39809-39821Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 21Mouro C. Bondon A. Jung C. Hui Bon Hoa G. De Certaines J.D. Spencer R.G. Simonneaux G. FEBS Lett. 1999; 455: 302-306Crossref PubMed Scopus (21) Google Scholar), and EPR studies (11Lipscomb J.D. Sligar S.G. Namtvedt M.J. Gunsalus I.C. J. Biol. Chem. 1976; 251: 1116-1124Abstract Full Text PDF PubMed Google Scholar, 22Shimada H. Nagano S. Ariga Y. Unno M. Egawa T. Hishiki T. Ishimura Y. Masuya F. Obata T. Hori H. J. Biol. Chem. 1999; 274: 9363-9369Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), Pdx binding has been shown to cause protein conformational changes that distort the P450-CAM heme structure and, especially, the Fe-S bond. In particular, Tosha et al. (20Tosha T. Yoshioka S. Takahashi S. Ishimori K. Shimada H. Morishima I. J. Biol. Chem. 2003; 278: 39809-39821Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) proposed that Pdx binding leads to structural changes that facilitate oxygen activation, and Unno et al. (18Unno M. Christian J.F. Sjodin T. Benson D.E. Macdonald I.D.G Sligar S.G. Champion P.M. J. Biol. Chem. 2002; 277: 2547-2553Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) suggested that the structural changes may correlate with H-bonding to the proximal Cys. Similarly, Nagano et al. (19Nagano S. Shimada H. Tarumi A. Hishiki T. Kimata-Ariga Y. Egawa T. Suematsu M. Park S.Y. Adachi S. Shiro Y. Ishimura Y. Biochemistry. 2003; 42: 14507-14514Crossref PubMed Scopus (50) Google Scholar) proposed that Pdx binding promotes electron donation from the proximal Cys to the iron-bound O2 to facilitate O-O bond cleavage. The role of hydrogen bonding in stabilizing heme-thiolate coordination in P450-CAM has been examined by Yoshioka et al. (23Yoshioka S. Tosha T. Takahashi S. Ishimori K. Hori H. Morishima I. J. Am. Chem. Soc. 2002; 124: 14571-14579Crossref PubMed Scopus (91) Google Scholar) and in a thiolate-ligated model system by Suzuki et al. (24Suzuki N. Higuchi T. Urano Y. Kikuchi K. Uekusa Y. Uchida T. Kitagawa T. Nagano T. J. Am. Chem. Soc. 1999; 121: 11571-11572Crossref Scopus (110) Google Scholar). In this report, we have used double mixing rapid scanning stopped-flow spectroscopy to monitor the reaction of oxyferrous P450-CAM with reduced Pdx in the presence or absence of substrate. The effect of oxidized Pdx on the stability of oxyferrous P450-CAM has also been studied. This study builds upon the highly demanding single mixing/single wavelength kinetics study of Brewer and Peterson (25Brewer C.B. Peterson J.A. J. Biol. Chem. 1988; 263: 791-798Abstract Full Text PDF PubMed Google Scholar), as well as the earlier work of Pederson et al. (26Pederson T.C. Austin R.H. Gunsalus I.C. Ullrich V. Roots I. Hildebrandt A. Estabrook R.W. Conney A.H. Microsomes and Drug Oxidations. Pergamon Press, Oxford, UK1977: 275-283Crossref Google Scholar) and Hui Bon Hoa et al. (27Hui Bon Hoa G. Begard E. Debey P. Gunsalus I.C. Biochemistry. 1978; 17: 2835-2839Crossref PubMed Scopus (19) Google Scholar), and provides considerable additional detail about the reaction between oxyferrous P-450 and reduced Pdx, including the effector roles of Pdx with P450-CAM. Two recent studies provide useful background for the reactions of reduced P-450 with dioxygen. Tosha et al. (28Tosha T. Yoshioka S. Hori H. Takahashi S. Ishimori K. Morishima I. Biochemistry. 2002; 41: 13883-13893Crossref PubMed Scopus (28) Google Scholar) recently investigated the roles of amino acids at the putative Pdx·P450-CAM interface, building on previous work by Unno et al. (29Unno M. Shimada H. Toba Y. Makino R. Ishimura Y. J. Biol. Chem. 1996; 271: 17869-17874Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Zhang et al. (30Zhang H. Gruenke L. Arscott D. Shen A. Kasper C. Harris D.L. Glavanovich M. Johnson R. Waskell L. Biochemistry. 2003; 42: 11594-11603Crossref PubMed Scopus (43) Google Scholar) have studied the analogous reduction of oxyferrous mammalian P-450-2B4 by its flavoprotein P-450 reductase. Materials and Sample Preparation—(1R)(+)-Camphor was purchased from Sigma, sodium dithionite was from Aldrich, and buffer chemicals were from Fisher. 5-exo-Hydroxy-(1R)-camphor was synthesized from (1R)-camphor following the procedure reported by Li et al. (31Li H. Narasimhulu S. Havran L.M. Winkler J.D. Poulos T.L. J. Am. Chem. Soc. 1995; 117: 6297-6299Crossref Scopus (64) Google Scholar) and was purified by silica gel column chromatography. P450-CAM and Pdx were individually overexpressed in Escherichia coli and purified as reported (32Unger B.P. Gunsalus I.C. Sligar S.G. J. Biol. Chem. 1986; 261: 1158-1163Abstract Full Text PDF PubMed Google Scholar). The ferric camphor-bound P450-CAM and oxidized Pdx concentrations were determined using ϵ391 = 102 mm-1 cm-1 and ϵ455 = 10.4 mm-1 cm-1, respectively (33Gunsalus I.C. Wagner G.C. Methods Enzymol. 1978; 52: 166-187Crossref PubMed Scopus (301) Google Scholar). The Pdx used had a ratio of A325/A280 ≥ 0.63 to establish purity. For stopped-flow experiments, P450-CAM and Pdx in 50 mm potassium phosphate, pH 7.4, 100 mm KCl, with or without 500 μm camphor, as required, were placed in tonometers either as separate solutions or mixed. Substrate-free P450-CAM was prepared by Sephadex G-25 chromatography in 50 mm MOPS, pH 7.0, to remove camphor, followed by a second G-25 column in 50 mm potassium phosphate, pH 7.4, 100 mm KCl (34Eble K.S. Dawson J.H. J. Biol. Chem. 1984; 259: 14389-14393Abstract Full Text PDF PubMed Google Scholar); stoichiometric camphor-bound P450-CAM was prepared by passing the protein through the latter G-25 column and then titrating back with just enough camphor to form the high spin ferric state. Protein solutions were made anaerobic by multiple cycles of alternately evacuating and flushing with oxygen-free argon at 25 °C over a period of 30 min. Reduced P450-CAM and Pdx were prepared by titration with microliter increments of a concentrated sodium dithionite stock solution until the ferric states were fully reduced as judged spectrally. Stopped-flow Experiments—Rapid scan stopped-flow experiments were carried out at 3-4 °C on a Hi-Tech Ltd. SF-61 DX2 instrument equipped for anaerobic work and with a Hi-Tech MG-6000 rapid scan diode array detector. Dead times were determined to be 1.5 ms. In single and double mixing experiments, numerous scans were recorded; the data displayed are representative. For double mixing experiments, a delay time of 100 ms was optimal for complete oxyferrous enzyme formation with essentially no autoxidation. The kinetic data were fit to various reaction models by the KinetAsyst program (Hi-Tech Ltd.) or by singular value decomposition and multiple component analysis to derive spectra of intermediates using the Specfit program from Spectrum Software Associates. Product Binding—The Kd value for binding 5-exo-hydroxy-camphor to ferric substrate-free P450-CAM was determined by Hill analysis of ligand titration data obtained by the addition of microliter aliquots of a concentrated stock solution of substrate to the substrate-free enzyme. Formation and Stability of Oxyferrous P450-CAM—The reaction of dioxygen at 4 °C with camphor-bound reduced P450-CAM occurs with a rate constant of ∼106 m-1 s-1 (data not shown). Thus, at a dioxygen concentration of 130 μm (aerobic buffer prepared at 20 °C (35Hitchman M.L. Measurement of Dissolved Oxygen. Wiley, Geneva, Switzerland1978: 7-33Google Scholar) mixed with anaerobic buffer), the rate would be about 130 s-1 (t½ = ∼5 ms). The UV-visible absorption spectrum of oxyferrous P450-CAM (Fig. 2) has Soret and visible region absorption peaks at 418 and 556 nm, respectively (419 and 554 nm at -30 °C in 65% ethylene glycol/phosphate buffer, pH 7.4) (36Sono M. Eble K.S. Dawson J.H. Hager L.P. J. Biol. Chem. 1985; 260: 15530-15535Abstract Full Text PDF PubMed Google Scholar). After formation of the oxyferrous species, essentially no spectral changes were observed for up to 60 s, as expected from the reported t½ for autoxidation of 25 min at this temperature (11Lipscomb J.D. Sligar S.G. Namtvedt M.J. Gunsalus I.C. J. Biol. Chem. 1976; 251: 1116-1124Abstract Full Text PDF PubMed Google Scholar). Substrate-free oxyferrous P450-CAM was also rapidly produced in a similar manner and likewise had UV-visible absorption peaks at 418 and 556 nm (data not shown). The t½ for autoxidation of substrate-free oxyferrous P450-CAM was determined to be 90 s at 4 °C, pH 7.4. In the double mixing experiments, the oxyferrous P450-CAM was allowed to form for ∼100 ms and then was mixed with the next reagent. Consequently, more than 99% of the P450-CAM was in the oxyferrous form at the time of the second mix. The Reaction of Camphor-bound Oxyferrous P450-CAM with Oxidized Pdx—In principle, oxidized Pdx should be incapable of reacting with oxyferrous cytochrome P-450 because, being in the oxidized state, it cannot provide an electron to reduce the oxyferrous enzyme. However, Lipscomb et al. (11Lipscomb J.D. Sligar S.G. Namtvedt M.J. Gunsalus I.C. J. Biol. Chem. 1976; 251: 1116-1124Abstract Full Text PDF PubMed Google Scholar) demonstrated nearly 30 years ago that oxidized Pdx could mediate the formation of up to one-half an equivalent of product from substrate-bound oxyferrous P450-CAM. The mechanism of this process has yet to be established. To quantify the kinetics of this effect, we examined the reaction of a 2-fold excess of oxidized Pdx with oxyferrous P450-CAM, first in the presence of a large excess of camphor and then in the presence of only a 2-fold excess of substrate. Dithionite-reduced ferrous P450-CAM in the presence of ∼100-fold excess of camphor was mixed, initially against aerobic buffer to create oxyferrous P450-CAM and then against a 2-fold excess of oxidized Pdx (Fig. 2A). The first trace is the spectrum of camphor-bound oxyferrous P450-CAM plus that of oxidized Pdx. Over the next 60 s, spectral changes yielded the spectrum of camphor-bound high spin ferric P450-CAM with peaks at 392, 539, and 561 nm plus minor contributions from Pdx. The t½ for this reaction is 10 s, 150-fold less than that for the formation of ferric camphor-bound P450-CAM from the oxyferrous enzyme (t½ = 25 min) in the absence of oxidized Pdx. The same experiment was repeated with oxyferrous P450-CAM prepared in the presence of only a 2-fold excess of substrate (Fig. 2B). The first spectral scan again shows the spectrum of oxyferrous P450-CAM. The last trace is the spectrum of a mixture of high and low spin state forms of ferric P450-CAM caused by the presence of the slight excess of camphor, plus contributions from Pdx. Normally, at these concentrations, one would expect that P450-CAM would be fully saturated with camphor and be high spin. However, the presence of Pdx weakens the binding of camphor (11Lipscomb J.D. Sligar S.G. Namtvedt M.J. Gunsalus I.C. J. Biol. Chem. 1976; 251: 1116-1124Abstract Full Text PDF PubMed Google Scholar). The t½ for breakdown of oxyferrous P450-CAM under these conditions was estimated to be 12 s. These two experiments (Fig. 2) reveal that the rate of conversion of oxyferrous to ferric P450-CAM is >100-fold enhanced (smaller t½) in the presence of oxidized Pdx. The rate enhancement is essentially the same whether the experiment is done in the presence of a large or small excess of camphor. This quantifies the dramatic extent to which oxidized Pdx decreases the t½ for formation of the ferric enzyme that Lipscomb et al. (11Lipscomb J.D. Sligar S.G. Namtvedt M.J. Gunsalus I.C. J. Biol. Chem. 1976; 251: 1116-1124Abstract Full Text PDF PubMed Google Scholar) had qualitatively reported. The Reaction of Camphor-bound Oxyferrous P450-CAM with Reduced Pdx in the Presence of Excess Camphor—This double mixing experiment involves the formation of camphor-bound oxyferrous P450-CAM in the first mixing step in which reduced P450-CAM is mixed with oxygenated buffer (Fig. 1, 3 → 4). After reacting for 100 ms, the second mixing step in the stopped-flow instrument introduces dithionite-reduced Pdx to initiate several steps converting 4 to 2 (Fig. 1). The camphor concentration (500 μm) was in large excess of the P450-CAM concentration (10 μm). Fig. 3 shows spectra recorded during the first 1000 ms of the reaction. The inset is an expanded view of the Soret absorption region. The first scan at 1.5 ms after the second mix is identified principally as the UV-visible absorption spectrum of oxyferrous P450-CAM, with a Soret absorption peak at 418 nm and a peak in the visible region at 554 nm. An initial increase in the intensity and a slight blue shift of the Soret absorption peak of the oxyferrous enzyme from 418 to ∼414 nm ensues during the first 17 ms, with an isosbestic point at 421 nm, and this is followed over the next 41 ms by a decrease in intensity and a further blue shift in peak position to a broad “double hump.” This process occurs with an isosbestic point at 405 nm. As will be discussed below, the rate of the second phase correlates with the rate reported by Brewer and Peterson (25Brewer C.B. Peterson J.A. J. Biol. Chem. 1988; 263: 791-798Abstract Full Text PDF PubMed Google Scholar) for the electron transfer from reduced Pdx to oxyferrous P450-CAM coupled with the formation of product. From this, we conclude that the protein is in the ferric state by the end of phase 2, and we thereby assign the broad double hump spectrum to a mixture of low spin and high spin ferric P450-CAM species. The third and slowest (k < 5s-1) phase seen in Fig. 3 occurs between 61 and 1000 ms. In this step, the Soret band is evolving with an isosbestic point at 408 nm into what is clearly high spin camphor-bound ferric P450-CAM. Singular value decomposition and global analysis of the data in Fig. 3 are shown in Fig. 4, which illustrates a fully resolved quite intense perturbed Soret peak at 413.5 nm (species B). The single peak in the visible region has decreased slightly in intensity and shifted from 554 to ∼548 nm. We refer to this species evolving in phase I as perturbed oxyferrous P450-CAM. This intermediate converts to C, the double-humped spectrum, which slowly converts to D, high spin camphor-bound ferric P450-CAM (Fig. 4). Because the perturbed oxyferrous enzyme Soret absorption peak is at nearly the same wavelength as that of ferric P450-CAM oxygen-donor ligand complexes (37Dawson J.H. Andersson L.A. Sono M. J. Biol. Chem. 1982; 257: 3606-3617Abstract Full Text PDF PubMed Google Scholar), we were initially concerned that the species at the end of the first phase might in fact be the product complex formed by binding of the alcohol oxygen of 5-exo-hydroxycamphor to ferric P450-CAM. However, as seen in the inset to Fig. 4, although the spectrum of 5-exo-hydroxy-camphor-bound ferric P450-CAM also has a Soret absorption peak at 413 nm, it has two distinct peaks in the visible region at 566 and 534 nm rather than the single peak seen for the perturbed oxyferrous species. Thus, despite the near coincidence in the position of their Soret absorption peaks, the overall absorption spectrum of 5-exo-hydroxycamphor-bound ferric P450-CAM is quite different from that of the perturbed oxyferrous P450-CAM·Pdx. These results demonstrate that the species formed at the end of phase 1 is not the product complex. To obtain more accurate and resolved kinetic data to use in determining rate constants for the first two phases, the experiment was repeated using single wavelength detection. Fig. 5 shows the spectral changes that occur at 418 nm for the first 100 ms. Data from this time period covers the first two phases of the reaction and could be fitted with a sum of two exponential functions. The first phase is characterized by a rate of ∼140 ± 14 s-1, and the second phase is characterized by a rate of ∼85 ± 8 s-1. Essentially identical rate constants were obtained at several different wavelengths (data not shown). The first rate corresponds to the conversion of oxyferrous P450-CAM to the perturbed oxyferrous intermediate. To assign the second rate constant, we turn to the work of Brewer and Peterson (25Brewer C.B. Peterson J.A. J. Biol. Chem. 1988; 263: 791-798Abstract Full Text PDF PubMed Google Scholar), who examined the reaction of reduced Pdx with oxyferrous P450-CAM under similar conditions to those described herein, but they used single mixing/single wavelength (420 nm) stopped-flow absorption spectroscopy, because of the limitations of the instrumentation available at that time. Only a single reaction phase with a rate of 60-100 s-1 was observed (from our data this would be expected, because the conversion of oxyferrous to the perturbed oxyferrous is essentially isosbestic at 420 nm). They assigned the rate to the electron transfer from reduced Pdx to oxyferrous P450-CAM on the basis of their careful parallel measurements of both the rate of electron transfer from reduced Pdx observed by freeze-quench EPR spectroscopy and the rate of product (5-exo-hydroxycamphor) formation, determined from chemical quench experiments. We therefore conclude that the rate of the second phase observed in Figs. 3 and 5 (∼85 s-1) likely corresponds to the rate of electron input from reduced Pdx to the perturbed oxyferrous intermediate and the formation of product. Additional support for this conclusion comes from the appearance during this phase of absorbance at ∼640 nm that is characteristic of high spin ferric P-450 (38Dawson J.H. Sono M. Chem. Rev. 1987; 87: 1255-1276Crossref Scopus (488) Google Scholar). The Reaction of Oxyferrous P450-CAM with Reduced Pdx in the Presence of Stoichiometric Camphor—We repeated the experiment with oxyferrous P450-CAM prepared with essentially only one equivalent of camphor bound to verify that camphor was being hydroxylated, as well as to separate out steps involved in releasing product and rebinding camphor. Camphor binds to ferric P450-CAM very tightly (Kd = ∼0.5 μm). This makes it possible to prepare a fully substrate-bound enzyme sample with essentially stoichiometrically added substrate. Reaction of the oxyferrous state of such a sample with reduced Pdx depletes the single equivalent of camphor, so that at the end of the experiment, the low spin ferric state (substrate-free) has formed. Fig. 6 displays UV-visible absorbance spectra recorded during the reaction. The formation of low spin substrate-free ferric P450-CAM is confirmed by the" @default.
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• W2074037790 title "Direct Observation of a Novel Perturbed Oxyferrous Catalytic Intermediate during Reduced Putidaredoxin-initiated Turnover of Cytochrome P-450-CAM" @default.
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