Abstract
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 Ca(2+) 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., FMNH(2), FAD, FADH., and FADH(2). The FMN/FMNH. couple is high potential ( approximately 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 approximately -270 mV in the presence of saturating arginine. Ca(2+)/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.
Highlights
Nitric oxide (NO) from arginine in a reaction requiring 3 electrons per NO, which are supplied to the catalytic center from
Potentiometric Titration of neuronal nitric-oxide synthase (nNOS) Holoenzyme—Changes in the redox state of cofactors are evaluated using their spectral features in the UV-visible region
The Soret band is sensitive to redox and spin state and in nNOS as isolated includes contributions from high and low spin heme
Summary
Vol 279, No 18, Issue of April 30, pp. 18759 –18766, 2004 Printed in U.S.A. Thermodynamics of Oxidation-Reduction Reactions in Mammalian Nitric-oxide Synthase Isoforms*. 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 oneelectron couples appear to be good enough reductants to participate in the catalytic cycle [20]. 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 [21,22,23] This is an important step in the catalytic cycle, since it makes the heme a competent acceptor of pyridine nucleotide-derived electrons. We have reported preliminary results from NOS holoenzymes (e.g. Refs. 28 and 29); here we describe important aspects of the redox behavior and coupling to protonation of three NOS mammalian isoforms
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