Thermodynamics of Oxidation-Reduction Reactions in Mammalian Nitric-oxide Synthase Isoforms

The three mammalian nitric-oxide synthases produce NO from arginine in a reaction requiring 3 electrons per NO, which are supplied to the catalytic center from NADPH through reductase domains incorporating FAD and FMN cofactors. The isoforms share a common reaction mechanism and requirements for reducing equivalents but differ in regulation; the endothelial and neuronal isoforms are controlled by calcium/calmodulin modulation of the electron transfer system, while the inducible isoform binds calmodulin at all physiological Ca2+ concentrations and is always on. The thermodynamics of electron transfer through the flavin domains in all three isoforms are basically similar. The major flavin states are FMN, FMNH·, FMNH2, FAD, FADH·, and FADH2. The FMN/FMNH· couple is high potential (∼100 mV) in all three isoforms and is unlikely to be catalytically competent; the other three flavin couples form a nearly isopotential group clustered around -250 mV. Reduction of the flavins by the pyridine nucleotide couple at -325 mV is thus moderately thermodynamically favorable. The ferri/ferroheme couple in all three isoforms is ∼-270 mV in the presence of saturating arginine. Ca2+/calmodulin has no effect on the potentials of any of the couples in endothelial nitric-oxide synthase (eNOS) or neuronal nitric-oxide synthase (nNOS). The pH dependence of the flavin couples suggests the presence of ionizable groups coupled to the flavin redox/protonation states. The three mammalian nitric-oxide synthases produce NO from arginine in a reaction requiring 3 electrons per NO, which are supplied to the catalytic center from NADPH through reductase domains incorporating FAD and FMN cofactors. The isoforms share a common reaction mechanism and requirements for reducing equivalents but differ in regulation; the endothelial and neuronal isoforms are controlled by calcium/calmodulin modulation of the electron transfer system, while the inducible isoform binds calmodulin at all physiological Ca2+ concentrations and is always on. The thermodynamics of electron transfer through the flavin domains in all three isoforms are basically similar. The major flavin states are FMN, FMNH·, FMNH2, FAD, FADH·, and FADH2. The FMN/FMNH· couple is high potential (∼100 mV) in all three isoforms and is unlikely to be catalytically competent; the other three flavin couples form a nearly isopotential group clustered around -250 mV. Reduction of the flavins by the pyridine nucleotide couple at -325 mV is thus moderately thermodynamically favorable. The ferri/ferroheme couple in all three isoforms is ∼-270 mV in the presence of saturating arginine. Ca2+/calmodulin has no effect on the potentials of any of the couples in endothelial nitric-oxide synthase (eNOS) or neuronal nitric-oxide synthase (nNOS). The pH dependence of the flavin couples suggests the presence of ionizable groups coupled to the flavin redox/protonation states. Nitric oxide (NO) 1The abbreviations used are: NO, nitric oxide; NOS, nitric-oxide synthase; eNOS, endothelial nitric-oxide synthase (NOSIII); iNOS, inducible nitric-oxide synthase (NOSII); nNOS, neuronal nitric-oxide synthase (NOSI); FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide. 1The abbreviations used are: NO, nitric oxide; NOS, nitric-oxide synthase; eNOS, endothelial nitric-oxide synthase (NOSIII); iNOS, inducible nitric-oxide synthase (NOSII); nNOS, neuronal nitric-oxide synthase (NOSI); FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide. is an important molecular messenger in a variety of signal transduction pathways (1Ignarro L.J. Buga G.M. Wood K.S. Byrns R.E. Chadhuri G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 9265-9269Google Scholar, 2Furchgott R.F. Vanhouette P.M. Vasodilation: Vascular Smooth Muscle, Peptides, Autonomic Nerves and Endothelium. Raven Press, New York1988: 401-404Google Scholar, 3Palmer R.M.J. Ferringe D.S. Moncada S. Nature. 1987; 327: 524-526Google Scholar, 4Garthwaite J. Charles S.L. Chess-Williams R. Nature. 1988; 336: 385-388Google Scholar). Nitric-oxide synthases (NOS) comprise a family of complex, modular enzymes, including three isoforms expressed in mammals. The endothelial and neuronal isoforms (eNOS and nNOS) are signal generators under the control of calcium/calmodulin (5Abu-Soud H.M. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10769-10772Google Scholar); a third isoform, iNOS, is induced during immune response (6Hauschildt S. Luckhoff A. Mulsch A. Kohler J. Bessler W. Busse R. Biochem. J. 1990; 270: 351-356Google Scholar, 7Knowles R.G. Merrett M. Salter M. Moncada S. Biochem. J. 1990; 270: 833-836Google Scholar, 8Curran R.D. J. Exp. Med. 1989; 170: 1769-1774Google Scholar, 9McCall T.B. Palmer R.M.J. Moncada S. Biochem. J. 1989; 262: 293-296Google Scholar) and produces much higher levels of NO even at basal levels of calcium. The generation of NO from arginine and oxygen by nitricoxide synthases requires the delivery of three pyridine nucleotide derived electrons to the catalytic site per mol of NO formed. The electron transfer system in endothelial nitric-oxide synthase (eNOS, NOSIII) utilizes one flavin mononucleotide (FMN) and one flavin adenine dinucleotide (FAD) cofactor (10Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Google Scholar, 11Mayer B. John M. Heinzel B. Werner E.R. Wachter H. Schultz G. Bohme E. FEBS Lett. 1991; 288: 187-191Google Scholar, 12McMillan K. Bredt D.S. Hirsch D.J. Snyder S.H. Clark J.E. Masters B.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11141-11145Google Scholar, 13Lamas S. Marsden P.A. Li G.K. Tempst P. Michel T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6348-6352Google Scholar, 14Lyons C.R. Orloff G.J. Cunningham J.M. J. Biol. Chem. 1992; 267: 6370-6374Google Scholar, 15Xie Q.W. Cho H.J. Calaycay J. Mumford R.A. Swiderek K.M. Lee T.D. Ding A. Troso T. Nathan C. Science. 1992; 256: 225-228Google Scholar) to deliver electrons derived from reduced nicotinamide dinucleotide phosphate (NADPH) to the site of oxygen chemistry, a protoporphyrin IX-derived heme with cysteinyl thiolate axial ligation (11Mayer B. John M. Heinzel B. Werner E.R. Wachter H. Schultz G. Bohme E. FEBS Lett. 1991; 288: 187-191Google Scholar, 12McMillan K. Bredt D.S. Hirsch D.J. Snyder S.H. Clark J.E. Masters B.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11141-11145Google Scholar, 16Stuehr D.J. Ikeda-Saito M. J. Biol. Chem. 1992; 267: 20547-20550Google Scholar, 17White K.A. Marletta M.A. Biochemistry. 1992; 31: 6627-6631Google Scholar). The electron transfer pathway is closely related to the NADPH P450 reductase/P450 system, and significant sequence homology exists between the flavoprotein binding domains in the two systems (10Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Google Scholar). The details of electron transfer differ significantly in FAD/FMN flavoprotein reductases related by homology and function. The two flavin cofactors have the capacity to accept four electrons. In mammalian P450 reductase, extensive studies have revealed that the system operates between the one and three electron reduced states (18Iyanagi T. Makino N. Mason H.S. Biochemistry. 1974; 13: 1701-1710Google Scholar, 19Munro A.W. Noble M.A. Robledo L. Daff S.N. Chapman S.K. Biochemistry. 2001; 40: 1956-1963Google Scholar). The FMN/FMNH· couple has a relatively high potential (∼-50 to -100 mV) and is not a kinetically competent reductant in the catalytic cycle. The FMNH·/FMNH2 and FAD/FADH· couples are intermediate in potential between the pyridine nucleotide pool and the physiological acceptor and are the primary functional one-electron couples in the catalytic cycle. The FADH·/FADH2 couple is too negative to be readily reduced by NADPH. Recently, the homologous reductase domains of cytochrome P450 BM3, which contains both flavin and P450 heme domains in a single polypeptide, have been shown to cycle primarily between the one- and two-electron reduced states; in this system, the order of the FMN potentials is reversed, and the FMN semiquinone stability constant is slightly less than unity. Both FMN one-electron couples appear to be good enough reductants to participate in the catalytic cycle (20Daff S.N. Chapman S.K. Turner K.L. Holt R.A. Govindaraj S. Poulos T.L. Munro A.W. Biochemistry. 1997; 36: 13816-13823Google Scholar). In enzymes of the P450 superfamily, it is well known that binding of “type I” substrates shifts the spin state equilibrium of ferriheme toward the high spin form, while simultaneously raising the midpoint potential (21Sligar S.G. Cinti D.L. Gibson G.G. Schenkman J.B. Biochem. Biophys. Res. Commun. 1979; 90: 925-932Google Scholar, 22Sligar S.G. Biochemistry. 1976; 15: 5399-5406Google Scholar, 23Gunsalus I.C. Sligar S.G. Biochimie (Paris). 1976; 58: 143-147Google Scholar). This is an important step in the catalytic cycle, since it makes the heme a competent acceptor of pyridine nucleotide-derived electrons. The phenomenon has been especially well studied in P450cam and has been discussed in terms of “spin state control” of redox equilibria (23Gunsalus I.C. Sligar S.G. Biochimie (Paris). 1976; 58: 143-147Google Scholar). An increase in the reduction of NOS ferriheme by NADPH has been reported after arginine addition (24Stuehr D.J. Abu-Soud H.M. Rousseau D.L. Feldman P.L. Wang J. Adv. Pharmacol. 1995; 34: 207-213Google Scholar), suggesting that a similar coupling of substrate binding and heme midpoint potential plays a role in that system as well. Previous studies of independently expressed reductase and oxygenase domains using potentiometric and stoichiometric titration have produced important information about the midpoint potentials of the heme and flavin components (25Noble M.A. Munro A.W. Rivers S.L. Robledo L. Daff S.N. Yellowlees L.J. Shimizu T. Sagami I. Guillemette J.G. Chapman S.K. Biochemistry. Scholar, M. Wang J. Biol. Chem. Scholar, A. Stuehr D.J. J. Chem. Scholar). of redox equilibria in enzymes and that at in domains heme and flavin potentials are have reported from NOS H.J. Guillemette J.G. D.L. J.B. Nitric Biol. Chem. and P.L. Nature. Scholar); important of the redox and coupling to of three NOS mammalian used for from and from and of to a at the expressed in and by a of and as M. J.B. Proc. Natl. Acad. Sci. U. S. A. 2001; Scholar). and of expressed in E. and as P. J. L.J. Masters B.S. Biochem. Biophys. Res. Commun. Scholar) that and using a at at concentrations on the of heme and of expressed in E. and as L.J. P. Masters B.S. Proc. Natl. Acad. Sci. U. S. A. 1995; Scholar), with the for under as by P.L. Scholar) using a titration with a by redox of known with an used in the system and of using from two heme redox states and flavin redox states. any are of the In is the is the and is the The of at is mV. The of the form of the redox states of the and flavins at for are the of components to the of the form the of all heme including and in equilibrium on the of redox in the is the potential to the as The midpoint potentials of the one-electron couples are by the and and are the midpoint potentials for the and one-electron of the FMN and In heme components that differ in and midpoint potential have been for are for the three FAD reduction states. Both the midpoint potentials and the at are on as redox state is of and states which as a single state in the potentiometric titration as as are in equilibrium on the of the of a titration at any is by the and the and at be by at which the of states are or by that the from one or is of The redox titration of the flavin cofactors at a single pH thus be in terms of flavin redox the flavins are well as electron of between the flavins requires but the be well The pH dependence of the system by of two states for flavin redox one state to the of the dependence of midpoint potential on pH by the pH and the midpoint potential at pH as T. H. T. J. Biol. Chem. 256: Scholar). The pH dependence of midpoint potentials is derived from ionizable groups with in the and reduced states. a one-electron couple with a single ionizable the pH dependence is of the a one-electron couple with two in the of the is of the The pH dependence which are of mV in the of a between the ionizable groups by the and be by the terms by and which the equilibrium constant for the of one group the other is of in the redox state of cofactors are using in the The of the is by the of the ferriheme at The is to redox and spin state and in as from high and spin flavins at and the is by the heme and the is as a on the The of is a of the reduction of FMN and Both flavin at and The at is by a transfer from high spin at a during an titration of the by a at the potential during the and are nearly for a and have been than with during a titration at pH The reduction of the heme the to with a midpoint potential of slightly mV. the potential is the flavin in the are and a reduced heme shown have from The for in the and potential are about in the to the at and The of and reduced are similar to the of and reduced enzymes and with concentrations of significantly the The of the is and in potential The with the information the at are to flavin and high spin information about all one-electron information be by of other but the semiquinone is in the The of the at during a titration is shown in with a which the of FAD and The makes of the known of the heme and flavin The and reduced flavins not in this and the heme is from the transfer of the high spin this to the of the states to The high potential is thus primarily to the for The flavin semiquinone with the potentials and for the and potential dependence of the flavin In a titration to the of the including the heme midpoint from FMN and FAD in that the concentrations and are to produce the with one flavin in or the potentials the in this but as the potential is heme and FAD to the slightly higher potential FMN semiquinone since the FMN is than the FAD the of the is the of the FMN/FMNH· and FADH·/FADH2 which are similar. the FMN is the to the of the heme transfer as FMN is formed. The potentials of the one-electron couples for are in I. The flavin couples are -250 mV for the FAD/FADH· couple mV for the FADH·/FADH2 couple mV for the FMNH·/FMNH2 couple and mV for the FMN/FMNH· couple The midpoint potential of the heme is mV as a single but be well by two components as potentials at pH for arginine and mammalian NOS and related systems potentials in the single the single to the are the with titration in the and reductase are from M.A. Munro A.W. Rivers S.L. Robledo L. Daff S.N. Yellowlees L.J. Shimizu T. Sagami I. Guillemette J.G. Chapman S.K. Biochemistry. in are in the presence of oxygenase are from M. Wang J. Biol. Chem. P450 reductase are from T. Makino N. Mason H.S. Biochemistry. 1974; 13: 1701-1710Google Scholar, and are from S.N. Chapman S.K. Turner K.L. Holt R.A. Govindaraj S. Poulos T.L. Munro A.W. Biochemistry. 1997; 36: 13816-13823Google mV mV mV mV mV mV in a are from the titration of the other The titration of the flavin around is shown in The are well by the heme and flavin potentials but the of the is not significant enough to the potentials of FMN and FAD from this of titration at is shown in The be in much the as the but the from the of the semiquinone are the flavin potentials are less by the the titration is with the for the flavin The are by including two heme one of which is significantly in potential than the single heme at other a and of the titration in the The heme titration has a midpoint mV the from the as a single as in this is at the of primarily of the of to the the potential in to other in the titration makes in the shown in a two significantly the The in the shown has a midpoint of -250 while the for about of the heme has a potential of be with potentials to mV for the potential of for the The heme of the is the of high spin ferriheme while the of the be the of the reduction of high spin and spin the titration in terms of a primarily high spin at -250 to mV with the and a primarily spin at which at to the that it with a high spin In the of arginine the titration of heme is and the midpoint potential is around mV. of arginine or are This be in the and and in the titration of the heme as components not The heme is primarily potential and spin and the of a variety of states not in In with with reductase domains (25Noble M.A. Munro A.W. Rivers S.L. Robledo L. Daff S.N. Yellowlees L.J. Shimizu T. Sagami I. Guillemette J.G. Chapman S.K. Biochemistry. Scholar) and with from no effect on heme or flavin potentials from the addition of Ca2+/calmodulin to the This is significant since NO is controlled by of electron transfer from NADPH to of of during potentiometric titration are similar to the shown in and have been to in nNOS, flavin are around in the potential is flavin in to be by of at This as the potential is ferriheme transfer to the in the it is the flavin in and as the heme is In the the during reduction is to the of the of the by a in the heme with the of flavin In the at is as a of The are similar to with The of the of FAD, and The at intermediate potentials is to FMN semiquinone since as in the FMN is than the FAD semiquinone the of the FMN/FMNH· and FADH·/FADH2 The of the has a as the potential is to the of the heme transfer as FMN is formed. The potentials of the one-electron couples for are in I. The flavin couples are mV for the FAD/FADH· couple mV for the FADH·/FADH2 couple mV for the FMNH·/FMNH2 couple and mV for the FMN/FMNH· couple The midpoint potential of the heme is mV. are with at other not of but of on the of the are of than the and of pH of titration of produced similar to the of shown in and have been by in form H.J. Guillemette J.G. D.L. J.B. Nitric Biol. Chem. Scholar). of the titration of the and of at three pH The be readily using the potentials shown in which are similar to the and The of is the of the potential at which be on the potential of the titration as the pH is from to with but suggesting the presence of a potential heme be readily produced at other for and pH a in the of the heme during the of a titration the in be for by the pH dependence of the midpoint potentials of the FAD couples in the from pH to pH Both one-electron couples are since the form of the in this is the the primary are FAD, FADH·, and FADH2. The pH dependence is slightly less than for a of per electron than the by including of and with the and with the and and with the reduced The semiquinone of the from the to the no of and the of the as the pH is but it is to from the from the pH at which to The of the since this in of the as a at pH and pH of the with the flavin are to be with the system but of groups to flavin redox/protonation reduced flavin of is to for the pH this the between and The reduced for that the of is the in all redox states; any is with the the pH dependence of the midpoint potentials of the FMN The pH dependence of the FMN couple is with a single of for the semiquinone and for the reduced in the of FAD, the with the semiquinone is not in a in this pH the couple to be pH This that at all pH in this no reduction to the a group with the flavin have a similar to that of the is that this couple to than the other flavin a with the potentials of the FMN couples and a mechanism related of to that the reductase domains of NOS, of control or to transfer electrons and that the couple is not of the catalytic cycle. The midpoint potential of the heme is in this even in arginine This that any pH dependence of arginine binding is of the redox state of the In the of arginine the heme titration is the potential is about mV at pH but is a high potential even in to which no arginine has been with nearly with eNOS, the heme and the FMN couples are and the flavin semiquinone is in the state in the pH from to in eNOS, the other three flavin couples are but in the pH dependence of couples is to to the The states are FAD, FADH·, FMN, FMNH·, and The for are not as but it is that at high pH three of the flavin couples are to that the states are the The of pH dependence for the FMN couple suggests the presence of a group coupled primarily to the than the of the a group be in the state but in the reduced and semiquinone states; this suggests in that is by to a titration of NOS isoforms information about the thermodynamics of electron transfer and and in addition about the coupling of redox to control of NO is through electron transfer from NADPH to heme through the flavin the thermodynamics of this is important in a of The of the three mammalian NOS isoforms to be similar. in the potential of the heme on arginine binding makes the step from to heme nearly and the heme couple and three of the four flavin couples form a nearly isopotential group slightly higher in potential than the pyridine that The is the high potential FMN/FMNH· which is about mV too to be an electron to The at about -325 is well to the three flavin couples in the isopotential and the high potential heme is no than mV in potential than the FMN couple which This slightly reaction be readily by oxygen or by NADPH. The potential on substrate binding is relatively with the in P450 systems (21Sligar S.G. Cinti D.L. Gibson G.G. Schenkman J.B. Biochem. Biophys. Res. Commun. 1979; 90: 925-932Google Scholar, 22Sligar S.G. Biochemistry. 1976; 15: 5399-5406Google Scholar, 23Gunsalus I.C. Sligar S.G. Biochimie (Paris). 1976; 58: 143-147Google Scholar). The potential in between and mV with saturating while shifts as as mV. The that the for arginine in reduced is than in the while a similar suggests that the arginine is in reduced than in This isoform in the between and than of the reduced between and on redox equilibria in NOS is a which be The heme potentials in NOS are slightly than in independently expressed oxygenase This is an effect of between the oxygenase and the reductase to a of the In nNOS, the potential of the arginine heme A. Stuehr D.J. J. Chem. Scholar) is mV higher than the for the The is much in the suggesting that the of by the reductase domains the state to the are no of arginine binding on the flavin midpoint by of NOS reductase the thermodynamics of the system are to P450 reductase than the reductase The NOS isoforms differ from P450 reductase in that the FADH·/FADH2 couple is nearly isopotential with the FAD/FADH· and FMNH·/FMNH2 couples as in I. in of reductase (25Noble M.A. Munro A.W. Rivers S.L. Robledo L. Daff S.N. Yellowlees L.J. Shimizu T. Sagami I. Guillemette J.G. Chapman S.K. Biochemistry. Scholar), no significant of calcium/calmodulin on any of the midpoint potentials This is with the of control through since the electron transfer be by than thermodynamics Scholar, K. B. P. L.J. Masters B.S. P. J. Biol. Chem. 1997; Scholar). The of the is not a significant in the modulation of electron transfer the major of the electron transfer are the shown to be and the between redox the heme and FMN of NOS be to using the of reductase significant to catalytic of electron Scholar), the FMN binding plays a role in enzymes homologous to the FAD and NADPH binding domains of NOS are reduced by or the are homologous to the NOS FMN binding domains and as an electron between FAD and redox components in other systems In the FMN binding of NOS as a from site on the to which it by a the FMN of the the heme site on the oxygenase to which it is through the calmodulin binding binding the this of the of the FMN binding from the An potential for the redox modulation of is the redox of groups in The pH dependence of and that the primary of the three redox states of both flavins are the between pH and the FMN is is not with the The of ionizable groups in the which are coupled to flavin redox/protonation state are especially through the pH midpoint potential of the FMN The used to produce the in the to the but pH dependence that differ slightly from of be produced by ionizable groups coupled to the redox states of the the other on the system, the of the semiquinone to pH the pH dependence the of a group which is in the FMN state and in the presence of the FMN This be to coupling of the ionizable group to FMN as from between an and the group in at one redox/protonation the that FMN is not to participate in this is of In the of the NOS isoforms during potentiometric a between the NOS reductase domains and P450 The between the isoforms is that in and control be to but isoform in or in the of between states with

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