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• W2029521537 abstract "Oxygen binding to the oxygenase domain of reduced endothelial nitric oxide synthase (eNOS) results in two distinct species differing in their Soret and visible absorbance maxima and in their capacity to exchange oxygen by CO. At 7 °C, heme-oxy I (with maxima at 420 and 560 nm) is formed very rapidly (kon ≈ 2.5·106 m–1·s–1) in the absence of substrate but in the presence of pterin cofactor. It is capable of exchanging oxygen with CO at –30 °C. Heme-oxy II is formed more slowly (kon ≈ 3·105 m–1·s–1) in the presence of substrate, regardless of the presence of pterin. It is also formed in the absence of both substrate and pterin. In contrast to heme-oxy I, it cannot exchange oxygen with CO at cryogenic temperature. In the presence of arginine, heme-oxy II is characterized by absorbance maxima near 432, 564, and 597 nm. When arginine is replaced by N-hydroxyarginine, and also in the absence of both substrate and pterin, its absorbance maxima are blue-shifted to 428, 560, and 593 nm. Heme-oxy I seems to resemble the ferrous dioxygen complex observed in many hemoproteins, including cytochrome P450. Heme-oxy II, which is the oxygen complex competent for product formation, appears to represent a distinct conformation in which the electronic configuration is essentially locked in the ferric superoxide complex. Oxygen binding to the oxygenase domain of reduced endothelial nitric oxide synthase (eNOS) results in two distinct species differing in their Soret and visible absorbance maxima and in their capacity to exchange oxygen by CO. At 7 °C, heme-oxy I (with maxima at 420 and 560 nm) is formed very rapidly (kon ≈ 2.5·106 m–1·s–1) in the absence of substrate but in the presence of pterin cofactor. It is capable of exchanging oxygen with CO at –30 °C. Heme-oxy II is formed more slowly (kon ≈ 3·105 m–1·s–1) in the presence of substrate, regardless of the presence of pterin. It is also formed in the absence of both substrate and pterin. In contrast to heme-oxy I, it cannot exchange oxygen with CO at cryogenic temperature. In the presence of arginine, heme-oxy II is characterized by absorbance maxima near 432, 564, and 597 nm. When arginine is replaced by N-hydroxyarginine, and also in the absence of both substrate and pterin, its absorbance maxima are blue-shifted to 428, 560, and 593 nm. Heme-oxy I seems to resemble the ferrous dioxygen complex observed in many hemoproteins, including cytochrome P450. Heme-oxy II, which is the oxygen complex competent for product formation, appears to represent a distinct conformation in which the electronic configuration is essentially locked in the ferric superoxide complex. Activation of molecular oxygen by nitric oxide synthase precedes NO 1The abbreviations used are: NO, nitric oxide; NOS, nitric-oxide synthase; eNOS, endothelial NOS; eNOSoxy, the oxygenase domain of recombinant bovine eNOS; nNOS, neuronal NOS; iNOS inducible NOS; NHA, NG-hydroxy-l-arginine; BH4, tetrahydrobiopterin ((6R)-5,6,7, 8-tetrahydro-6-(L-erythro-1′,2′-dihydroxypropyl)pterin); ABH4 (4-amino-BH4), 4-amino-tetrahydrobiopterin ((6R)-2,4-diamino-5,6,7,8-tetrahydro-6-(L-erythro-1′,2′-dihydroxypropyl)pteridine); BH2, 7,8-dihydro-l-biopterin; CHAPS, 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate. 1The abbreviations used are: NO, nitric oxide; NOS, nitric-oxide synthase; eNOS, endothelial NOS; eNOSoxy, the oxygenase domain of recombinant bovine eNOS; nNOS, neuronal NOS; iNOS inducible NOS; NHA, NG-hydroxy-l-arginine; BH4, tetrahydrobiopterin ((6R)-5,6,7, 8-tetrahydro-6-(L-erythro-1′,2′-dihydroxypropyl)pterin); ABH4 (4-amino-BH4), 4-amino-tetrahydrobiopterin ((6R)-2,4-diamino-5,6,7,8-tetrahydro-6-(L-erythro-1′,2′-dihydroxypropyl)pteridine); BH2, 7,8-dihydro-l-biopterin; CHAPS, 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate. biosynthesis by a yet incompletely known reaction mechanism. A study of the steps following oxygen binding and NO formation is important since NO is a mediator of a wide range of physiological and pathophysiological processes in humans and other mammals (1Ignarro L.J. Hypertension. 1990; 16: 477-483Crossref PubMed Scopus (422) Google Scholar, 2Bredt D.S. Snyder S.H. Annu. Rev. Biochem. 1994; 63: 175-195Crossref PubMed Scopus (2131) Google Scholar, 3Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar, 4Hemmens B. Mayer B. Methods Mol. Biol. 1998; 100: 1-32PubMed Google Scholar, 5Marletta M.A. Hurshman A.R. Rusche K.M. Curr. Opin. Chem. Biol. 1998; 2: 656-663Crossref PubMed Scopus (203) Google Scholar, 6Murphy M.P. Biochim. Biophys. Acta. 1999; 1411: 401-414Crossref PubMed Scopus (362) Google Scholar). The formation of NO is catalyzed by nitric oxide synthases (NOS; EC 1.14.13.39) via a two-step mechanism. The substrate, l-Arg, is first converted to NG-hydroxy-l-Arg (NHA) at the heme active site. In the second step, NHA is further oxidized to NO and citrulline (3Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar). In both steps, oxygen binding occurs after a one-electron reduction of the ferric heme. Prior to product formation, an electron stemming from tetrahydrobiopterin (BH4) then further reduces the ferrous oxygen complex. Despite much effort, the reaction mechanism of these steps is still unclear. Even the spectral properties of the oxyferrous complex are not yet defined; conflicting observations report absorbance maxima differing by up to 15 nm. The origin of these differences is unknown. The strongest differences are those between observations at cryogenic temperatures yielding maxima in the 417–419-nm region (7Bec N. Gorren A.C. Voelker C. Mayer B. Lange R. J. Biol. Chem. 1998; 273: 13502-13508Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 8Ledbetter A.P. McMillan K. Roman L.J. Masters B.S. Dawson J.H. Sono M. Biochemistry. 1999; 38: 8014-8021Crossref PubMed Scopus (56) Google Scholar, 9Gorren A.C. Bec N. Schrammel A. Werner E.R. Lange R. Mayer B. Biochemistry. 2000; 39: 11763-11770Crossref PubMed Scopus (74) Google Scholar) and observations at higher temperatures by rapid scan techniques yielding considerably red-shifted maxima (430–432 nm) (10Abu-Soud H.M. Gachhui R. Raushel F.M. Stuehr D.J. J. Biol. Chem. 1997; 272: 17349-17353Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 11Sato H. Sagami I. Daff S. Shimizu T. Biochem. Biophys. Res. Commun. 1998; 253: 845-849Crossref PubMed Scopus (37) Google Scholar, 12Boggs S. Huang L. Stuehr D.J. Biochemistry. 2000; 39: 2332-2339Crossref PubMed Scopus (65) Google Scholar, 13Couture M. Stuehr D.J. Rousseau D.L. J. Biol. Chem. 2000; 275: 3201-3205Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 14Wei C.C. Wang Z.Q. Wang Q. Meade A.L. Hemann C. Hille R. Stuehr D.J. J. Biol. Chem. 2001; 276: 315-319Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). However, this distinction is not clear-cut; in some cases, low wavelength maxima are also found by rapid scan spectroscopy (11Sato H. Sagami I. Daff S. Shimizu T. Biochem. Biophys. Res. Commun. 1998; 253: 845-849Crossref PubMed Scopus (37) Google Scholar), and high wavelength maxima are also found by low temperature UV-visible spectroscopy (9Gorren A.C. Bec N. Schrammel A. Werner E.R. Lange R. Mayer B. Biochemistry. 2000; 39: 11763-11770Crossref PubMed Scopus (74) Google Scholar). To clarify this confusing situation, we reanalyzed the temporal evolution of the spectral changes occurring upon oxygen binding to reduced eNOS oxygenase domain by rapid scanning stopped-flow, assisted by global fit analysis. The present spectral and kinetic data show that, depending on experimental conditions such as temperature and presence of substrate and pteridine cofactor, two different heme-oxy complexes are formed. These complexes are further distinguished by their ability to exchange oxygen by CO at –30 °C. The implication of two different oxygen complexes in NOS distinguishes this enzyme from other heme-thiolate-containing proteins. In addition, the dependence of these oxy-complexes on other reaction partners may be important in the mechanism of uncoupling of NADPH oxidation from product formation. Therefore, formation, stability, and decay of the oxygen complexes were followed within both eNOS reaction cycles (using l-Arg and NHA as substrate) and in the presence of different BH4 analogues. Materials—The oxygenase domain of bovine endothelial NOS (eNOSoxy) was expressed in and purified from Escherichia coli (15Schmidt P.P. Lange R. Gorren A.C. Werner E.R. Mayer B. Andersson K.K. J. Biol. Inorg. Chem. 2001; 6: 151-158Crossref PubMed Scopus (95) Google Scholar). All chemicals were from Sigma except for pteridines (Schircks Laboratories, Jona, Switzerland), argon, O2, and CO (Aga, Toulouse, France). Sample Preparation—Samples were prepared as described (7Bec N. Gorren A.C. Voelker C. Mayer B. Lange R. J. Biol. Chem. 1998; 273: 13502-13508Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 9Gorren A.C. Bec N. Schrammel A. Werner E.R. Lange R. Mayer B. Biochemistry. 2000; 39: 11763-11770Crossref PubMed Scopus (74) Google Scholar, 16Lange R. Bec N. Anzenbacher P. Munro A.W. Gorren A.C. Mayer B. J. Inorg. Biochem. 2001; 87: 191-195Crossref PubMed Scopus (12) Google Scholar). Briefly, NOS samples (2–6 μm eNOSoxy) in KCEM buffer (50 mm KPi at pH 7.5, 1 mm CHAPS, 0.5 mm EDTA, 1 mm 2-mercaptoethanol), in the presence or absence of substrates (1 mm NHA or 0.5 mm l-Arg) and pteridines (100 μm) were deoxygenated under argon atmosphere and then reduced by sodium dithionite (1 mm final concentration) at 15 °C. CHAPS (which does not affect the spectral properties of the enzyme) was used to maintain the structural and functional integrity of NOS (17Klatt P. Pfeiffer S. List B.M. Lehner D. Glatter O. Bachinger H.P. Werner E.R. Schmidt K. Mayer B. J. Biol. Chem. 1996; 271: 7336-7342Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). For stopped-flow experiments, O2-saturated stock solutions were prepared by bubbling KCEM buffer with O2 for 45 min. The concentration of oxygen in these solutions was assumed to be 1 mm at 25 °C. Argon-saturated solutions were prepared by the same procedure. Stopped-flow Spectroscopy—Rapid-scanning experiments were performed at 7 °C using an SFM 300 BioLogic Instrument (Grenoble, France) stopped-flow apparatus equipped with a J&M TIDAS diode array detector (MCS/16-TSPEC/500kHz). Anaerobic solutions of ferrous eNOS oxygenase domain were mixed with equal volumes of oxygen-containing buffer. The final concentration of oxygen was varied by the use of three independent mixing syringes: one containing the enzyme, one containing the oxygen-saturated buffer, and one containing the argon-saturated buffer. The mixing dead time was 0.3 ms. After each injection, two hundred spectra from 350 to 700 nm were collected with an acquisition time of 2.5 ms. Rapid-scanning data were compiled and fitted to different reaction models using the Specfit global analysis program (provided by the instrument manufacturer), which evaluates the number of different enzyme species, their individual spectra, and the concentration of each species as a function of time, as well as the rate constant for each transition. In some experiments where the kinetics of formation of the oxygenated eNOSoxy complexes were too rapid to be detected with the diode array (lower limit of integration time: 2.5 ms), the kinetics were followed at a single wavelength (414 nm) with a PMS-250 photomultiplier (BioLogic Science Instruments), which allowed the kinetic resolution of reactions occurring within a couple of milliseconds. All experiments were performed at least twice, with different batches of eNOSoxy. The kinetics of formation and decay of oxygen complexes were not affected by the presence of excess dithionite; control experiments using different concentrations of dithionite (0.5–5 mm) and of oxygen (0.1–0.5 mm) did not reveal any effect of dithionite on the kinetics. Low Temperature Optical Spectroscopy—Low temperature UV-visible absorption spectra of the reaction between reduced eNOSoxy and O2 or CO were recorded with a Cary 3E (Varian, Palo Alto, CA) spectrophotometer, adapted for low temperature studies, according to previously published procedures (7Bec N. Gorren A.C. Voelker C. Mayer B. Lange R. J. Biol. Chem. 1998; 273: 13502-13508Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 9Gorren A.C. Bec N. Schrammel A. Werner E.R. Lange R. Mayer B. Biochemistry. 2000; 39: 11763-11770Crossref PubMed Scopus (74) Google Scholar, 18Gorren A.C. Bec N. Lange R. Mayer B. Methods Enzymol. 2002; 353: 114-121Crossref PubMed Scopus (9) Google Scholar). To avoid freezing, the experiments were carried out in a 1:1 (v/v) mixture of aqueous buffer/ethylene glycol. The organic cosolvent did not change significantly the spectral properties of NOS, it did not induce a transition to the P420 state, and in its presence, the enzyme was still active in a standard assay (19Mayer B. Klatt P. Werner E.R. Schmidt K. Neuropharmacology. 1994; 33: 1253-1259Crossref PubMed Scopus (137) Google Scholar). Detection of Two Spectrally Distinct Oxygen Complexes— Depending on experimental conditions, rapid mixing of reduced eNOSoxy with oxygen-containing buffer resulted in two different spectral species. As shown in Fig. 1, one species is characterized by a Soret absorbance maximum of 420 nm and a single visible band at 560 nm, the other one is characterized by a considerably red-shifted Soret maximum (428 nm) and by two visible bands (560 and 595 nm). For simplicity, we denote the first one heme-oxy I, and we denote the latter one heme-oxy II. Inspection of Table I shows that heme-oxy I is formed in the absence of substrate, regardless of the nature of the pterin cofactor. In contrast, heme-oxy II is formed in the presence of substrate (l-Arg or NHA), irrespective of the pterin cofactor. In addition, heme-oxy II is also formed when both substrate and pterin cofactor are absent. A closer analysis of Table I reveals small spectral differences of heme-oxy II when formed in the presence of l-Arg or NHA. With l-Arg, a Soret maximum is found near 432 nm, and α/β bands are found at 564 and 597 nm. With NHA, the Soret band culminates at 428 nm, and the α/β bands culminate at 560 and 593 nm. Interestingly, when the experiments were carried out at –30 °C, under some conditions, different results were obtained. For instance, at low temperature, the presence of NHA and absence of pterin resulted in an oxygen complex of heme-oxy I spectral characteristics.Table ISpectral properties of heme-dioxy complexes detected in single turnover reactions of eNOS oxygenase domain with oxygenSubstratePterinλmaxIntermediateSoretα,βnmBH4421560heme-oxy I4-amino-BH4420561heme-oxy IBH2421560heme-oxy IArg433565/595heme-oxy IIArgBH4432563/597heme-oxy IIArg4-amino-BH4432565/600heme-oxy IIArgBH2431563/598heme-oxy IINHA428 (419)560/595 (560)heme-oxy II (I)NHABH4428560/590heme-oxy IINHA4-amino-BH4429 (425)560/593 (557/595)heme-oxy IINHABH2429 (426)560/594 (557/595)heme-oxy II-428560/595heme-oxy II Open table in a new tab Occurrence of Heme-oxy II within the Second Reaction Cycle— Rapid mixing of reduced eNOSoxy with O2-saturated buffer in the presence of BH4 and NHA resulted in a gradual red shift of the Soret band from 414 to ∼440 nm followed by a shift in the opposite direction finally yielding ferric heme (394 nm) (Fig. 2). The temporal sequence of spectra was best fitted to a sequential model with three monophasic transitions between four distinct species. The four spectral components revealed by global analysis were ferrous eNOSoxy (λmax 414 nm), heme-oxy II (characterized by absorbance peaks at 428, 560, and a shoulder at 600 nm), the Fe(III)-NO complex with λmax(Soret) 438 nm and λmax(α,β) 547/583 nm, and high spin ferric heme (λmax 394 nm). The Soret and visible absorbance features of the transient species after O2 addition and of ferric-NO match those of oxygenated (FeIIO2) and FeIIINO complexes of iNOSoxy, nNOSoxy, or eNOSoxy characterized previously at 10 °C (10Abu-Soud H.M. Gachhui R. Raushel F.M. Stuehr D.J. J. Biol. Chem. 1997; 272: 17349-17353Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 12Boggs S. Huang L. Stuehr D.J. Biochemistry. 2000; 39: 2332-2339Crossref PubMed Scopus (65) Google Scholar, 20Wang Z.Q. Wei C.C. Stuehr D.J. J. Biol. Chem. 2002; 277: 12830-12837Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 21Wei C.C. Wang Z.Q. Arvai A.S. Hemann C. Hille R. Getzoff E.D. Stuehr D.J. Biochemistry. 2003; 42: 1969-1977Crossref PubMed Scopus (54) Google Scholar). The transient occurrence of heme-oxy II and Fe(III)-NO also appears evident from the time course of the concentration of the four species, which was deduced by global fit analysis (Fig. 2C). As shown in Fig. 3, a similar spectral evolution was observed when BH4 was replaced by ABH4, except that no Fe(III)-NO complex was observed, which is in line with recent observations (22Hurshman A.R. Krebs C. Edmondson D.E. Marletta M.A. Biochemistry. 2003; 42: 13287-13303Crossref PubMed Scopus (52) Google Scholar). Therefore, in this case, the reaction contained only two steps: conversion of ferrous heme to heme-oxy II followed by its decay to ferric heme. Clear isosbestic points characterized both processes. Kinetic Analysis of Heme-oxy I and Heme-oxy II Formation— The formation of heme-oxy I was too fast for a precise determination of rate constants from rapid scanning stopped-flow data. Therefore, we used single wavelength stopped-flow. Fig. 4A is an example for the kinetic traces observed. Heme-oxy I forms within a few milliseconds and decays within 100 ms. In contrast, the kinetics of formation and decay of heme-oxy II were much slower and could be analyzed adequately from rapid scan data. Fig. 4B shows the dependence of the kobs of the formation of heme-oxy II as a function of oxygen concentration. Clearly, a linear relationship was observed, indicating a pseudo-first order reaction. Accordingly, kon and koff were determined using Equation 1. kobs=koff+kon×[O2](Eq. 1) The kinetic rate constants under the different experimental conditions are listed in Table II. In most cases, the formation of heme-oxy II was irreversible as evidenced by a koff value close to 0. 2With BH4 analogues or in the absence of pterins, a negative intercept value was estimated from plots of kobs versus O2 concentration for eNOSoxy in the presence of substrates. To indicate the irreversible process of O2 binding under these conditions, we tabulated the O2 dissociation constant value as 0. Exceptions were the reactions in the presence of substrate and BH4 (koff = 23 ± 1 s–1) and in the presence of NHA and BH2 (koff = 9.7 s–1). To enable a comparison of the formation rates of heme-oxy I and heme-oxy II, the experimental values of kobs for heme-oxy I were converted into second order rate constants, using a single concentration of oxygen (150 μm). At higher concentrations, oxygen binding occurred within the mixing time, precluding analysis of the [O2] dependence. For both heme-oxy I and heme-oxy II, the decay rate constants (reflecting the transition to ferric high spin or to Fe(III)-NO) were independent of oxygen concentration, i.e. the decay was a first order reaction.Table IIKinetic parameters of the intermediates after oxygen addition to reduced eNOS oxygenase domain in the presence and absence of substrates and pterinsConditionsHeme-oxyFeIII-NO (kdecay)konkoffkdecaymm-1·s-1s-1s-1s-1NHA+BH43422214.52.5+4-amino-BH427200.086+BH22279.70.134no pterin14500.358Arginine+BH4265246.2+4-amino-BH441000.0536+BH245200.181no pterin33801.49No substrate/pterin2480aPseudo first order rates estimated in the presence of 0.15 mm O2.NDbND, not determined.60/2.1BH42333aPseudo first order rates estimated in the presence of 0.15 mm O2.NDbND, not determined.144/184-amino-BH42758aPseudo first order rates estimated in the presence of 0.15 mm O2.NDbND, not determined.27/0.3BH22600aPseudo first order rates estimated in the presence of 0.15 mm O2.NDbND, not determined.34/0.3a Pseudo first order rates estimated in the presence of 0.15 mm O2.b ND, not determined. Open table in a new tab A particular situation was encountered when both substrate and pterin cofactor were absent. In this case, the starting spectrum recorded after 2.5 ms of mixing displayed essentially identical spectral features of heme-oxy II. This transient species decayed to form ferric eNOSoxy, as judged by a shift of the Soret band to a broad spectrum centered at 410 nm and build-up of visible absorbance at 630 nm (data not shown). Using single-wavelength stopped-flow, we showed that the formation kinetics of the oxygenated intermediate were very fast (a couple of milliseconds), similar to the formation of heme-oxy I (Table II). Kinetic Effects of Pterins and Substrates—As shown in Table II, the kinetics of heme-oxy I formation did not depend significantly on the nature of the pterin cofactor. In contrast, its decay rate was about 5 times higher in the presence of BH4 with respect to other pterins. Similarly, the kon rate of heme-oxy II did not vary strongly under the different conditions. However, its decay rate was tremendously (more than 100-fold) enhanced in the presence of BH4. Similar tendencies were observed for both substrates (l-Arg and NHA). Oxygen Exchange by CO—To further characterize heme-oxy I and heme-oxy II, we investigated their reactivity versus CO. To do that, we chose conditions at –30 °C where heme-oxy I or II were predominant and bubbled CO through the sample immediately after formation of the oxygen complex. As shown in Fig. 5, the two oxygen complexes behaved differently. Heme-oxy I transformed to a spectrum with contributions from two compounds; the Soret region exhibited maxima at 396 and 444 nm. The 396-nm peak most likely reflects the oxidized form, whereas the latter was typical of thiolate-ligated ferrous-CO heme (λmax 444 nm). In contrast, heme-oxy II did not exchange CO; its 427-nm Soret maximum shifted without intermediate to 395 nm, indicating a decay of heme-oxy II to ferric high spin. Are Heme-oxy I and Heme-oxy II Distinct Intermediates?— One of the most salient observations in the present study is the detection of intermediates in the oxygenation of reduced eNOSoxy of different spectral and kinetic properties (heme-oxy I and heme-oxy II). The question now is whether these two spectral forms represent distinct reaction intermediates. There are prior indications for the existence of different NOS oxygen complexes. Intermediates with absorbance maxima at 417–419 nm (of heme-oxy type I) have been observed in several low temperature optical studies under a variety of conditions (7Bec N. Gorren A.C. Voelker C. Mayer B. Lange R. J. Biol. Chem. 1998; 273: 13502-13508Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 8Ledbetter A.P. McMillan K. Roman L.J. Masters B.S. Dawson J.H. Sono M. Biochemistry. 1999; 38: 8014-8021Crossref PubMed Scopus (56) Google Scholar, 9Gorren A.C. Bec N. Schrammel A. Werner E.R. Lange R. Mayer B. Biochemistry. 2000; 39: 11763-11770Crossref PubMed Scopus (74) Google Scholar). A maximum at 417 nm was also found by rapid scan spectroscopy for full-length nNOS in the absence of substrate and BH4 (11Sato H. Sagami I. Daff S. Shimizu T. Biochem. Biophys. Res. Commun. 1998; 253: 845-849Crossref PubMed Scopus (37) Google Scholar). However, most stopped-flow/rapid scan studies yielded intermediates with Soret maxima at 427–431 nm, i.e. of heme-oxy type II (10Abu-Soud H.M. Gachhui R. Raushel F.M. Stuehr D.J. J. Biol. Chem. 1997; 272: 17349-17353Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 11Sato H. Sagami I. Daff S. Shimizu T. Biochem. Biophys. Res. Commun. 1998; 253: 845-849Crossref PubMed Scopus (37) Google Scholar, 12Boggs S. Huang L. Stuehr D.J. Biochemistry. 2000; 39: 2332-2339Crossref PubMed Scopus (65) Google Scholar, 14Wei C.C. Wang Z.Q. Wang Q. Meade A.L. Hemann C. Hille R. Stuehr D.J. J. Biol. Chem. 2001; 276: 315-319Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 20Wang Z.Q. Wei C.C. Stuehr D.J. J. Biol. Chem. 2002; 277: 12830-12837Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 21Wei C.C. Wang Z.Q. Arvai A.S. Hemann C. Hille R. Getzoff E.D. Stuehr D.J. Biochemistry. 2003; 42: 1969-1977Crossref PubMed Scopus (54) Google Scholar, 23Abu-Soud H.M. Ichimori K. Presta A. Stuehr D.J. J. Biol. Chem. 2000; 275: 17349-17357Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 24Wei C.C. Wang Z.Q. Hemann C. Hille R. Stuehr D.J. J. Biol. Chem. 2003; 278: 46668-46673Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). These spectral differences cannot be exclusively explained by different experimental temperature or the presence of cryogenic cosolvent (8Ledbetter A.P. McMillan K. Roman L.J. Masters B.S. Dawson J.H. Sono M. Biochemistry. 1999; 38: 8014-8021Crossref PubMed Scopus (56) Google Scholar, 9Gorren A.C. Bec N. Schrammel A. Werner E.R. Lange R. Mayer B. Biochemistry. 2000; 39: 11763-11770Crossref PubMed Scopus (74) Google Scholar, 11Sato H. Sagami I. Daff S. Shimizu T. Biochem. Biophys. Res. Commun. 1998; 253: 845-849Crossref PubMed Scopus (37) Google Scholar, 12Boggs S. Huang L. Stuehr D.J. Biochemistry. 2000; 39: 2332-2339Crossref PubMed Scopus (65) Google Scholar, 25Bec N. Gorren A.F.C. Mayer B. Schmidt P.P. Andersson K.K. Lange R. J. Inorg. Biochem. 2000; 81: 207-211Crossref PubMed Scopus (59) Google Scholar) since red-shifted oxygen complexes with absorbance maxima up to 432 nm have been reported in low temperature studies as well (9Gorren A.C. Bec N. Schrammel A. Werner E.R. Lange R. Mayer B. Biochemistry. 2000; 39: 11763-11770Crossref PubMed Scopus (74) Google Scholar). Some of the reported spectral discrepancies may have been artificial in those cases, when incomplete formation of a 428-/432-nm intermediate resulted in an apparent maximum at lower wavelength, as discussed previously (9Gorren A.C. Bec N. Schrammel A. Werner E.R. Lange R. Mayer B. Biochemistry. 2000; 39: 11763-11770Crossref PubMed Scopus (74) Google Scholar). However, the extreme stability of some of the observed intermediates appears to preclude such an explanation in many instances. The presence and identity of substrate and pteridines may also have affected the spectral properties of the oxyferrous complexes (9Gorren A.C. Bec N. Schrammel A. Werner E.R. Lange R. Mayer B. Biochemistry. 2000; 39: 11763-11770Crossref PubMed Scopus (74) Google Scholar, 24Wei C.C. Wang Z.Q. Hemann C. Hille R. Stuehr D.J. J. Biol. Chem. 2003; 278: 46668-46673Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), in line with prior observations for cytochrome P450 (26Tuckey R.C. Kamin H. J. Biol. Chem. 1982; 257: 9309-9314Abstract Full Text PDF PubMed Google Scholar). Several observations in the present study argue in favor of two distinct heme-oxy complexes: i) the observed spectra can be clearly distinguished in two groups with well defined properties in the Soret and α,β regions; ii) the effects of substrate and pterines on the spectra follow regular patterns; (iii) although in most cases the red-shifted spectra exhibited much greater stability than the blue-shifted intermediates, this property was not universal since a short-lived 428-nm intermediate was formed in the absence of substrate and pterin, which rules out that the blue species are experimental artifacts; iv) at low temperature, formation of heme-oxy I is reversible, and oxygen can be replaced by CO. This is not the case for heme-oxy II. Particularly, the different reactivity versus CO constitutes a strong argument for the existence of two different oxygen complexes. Nature of Heme-oxy I and II—All spectral species that were formed in the absence of pteridines and in the presence of BH2 must have been in the same [Fe·O2]2+ redox state since under those conditions, further reduction of the oxy-complex was impossible. In those instances, where a second electron transfer was possible (l-Arg/NHA+BH4/ABH4), a different interpretation (for heme-oxy II) would be theoretically possible but highly improbable, in view of the near identity with the spectra obtained under non-productive (BH2 or absence of pterin) conditions (Table I). Indeed, EPR spectra corresponding to the oxy-complexes observed at low temperature showed no trace of a ferrous-superoxy or ferri-peroxy complex (27Sorlie M. Gorren A.C. Marchal S. Shimizu T. Lange R. Andersson K.K. Mayer B. J. Biol. Chem. 2003; 278: 48602-48610Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). We conclude that all spectral species that were designated heme-oxy I and II by us in the present study were isoelectronic, formally presented by the [Fe·O2]2+ redox state. The spectral properties of heme-oxy I are reminiscent of the oxy-complexes of other hemoproteins, such as cytochrome P450 and globins (28Gunsalus I.C. Meeks J.R. Lipscomb J.D. Debrunner P.G. Münck E. Hayaishi O. Molecular Mechanisms of Oxygen Activation. Academic Press, Orlando, FL1974: 559-613Google Scholar). In those cases, there is spectroscopic evidence (29Sharrock M. Debrunner P.G. Schulz C. Lipscomb J.D. Marshall V. Gunsalus I.C. Biochim. Biophys. Acta. 1976; 420: 8-26Crossref PubMed Scopus (123) Google Scholar) that the predominant electronic state is the ferric superoxide (Fe3+−O2⋅¯), suggesting a similar electronic structure for heme-oxy I. However, this species must still have a considerable contribution of the ferrous-dioxygen (Fe2+-O2) mesomer since the complex readily exchanged O2 for CO, as was also observed for cytochromes P450 and globins (30Sono" @default.
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