Comparative Effects of Substrates and Pterin Cofactor on the Heme Midpoint Potential in Inducible and Neuronal Nitric Oxide Synthases

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• 作者：Anthony Presta ; Anne M. Weber-Main ; Marian T. Stankovich ; and Dennis J. Stuehr
• 刊名：Journal of the American Chemical Society
• 出版年：1998
• 出版时间：September 23, 1998
• 年：1998
• 卷：120
• 期：37
• 页码：9460 - 9465
• 全文大小：157K
• 年卷期：v.120,no.37(September 23, 1998)
• ISSN：1520-5126

文摘

The nitric oxide synthases (NOS) are heme-containing enzymes responsible for catalyzing the five-electron oxidation of a guanidino nitrogen of L-arginine to produce the free radical nitric oxide. The bindingsites of the heme group, as well as of the L-arginine substrate and tetrahydrobiopterin cofactor, are locatedwithin the oxygenase domain of the NOS enzymes. Reduction of the heme is the first committed step incatalysis, as this allows for binding and activation of molecular oxygen, followed by oxidative attack on theL-arginine substrate. As with heme groups in other enzymes, the electronic properties of the NOS heme aremodified by substrate and cofactor binding in its vicinity. Here we present the first quantitative thermodynamicdata of the NOS heme with the determination of the heme midpoint reduction potentials for the neuronal NOSand inducible NOS oxygenase domains. In the absence of L-arginine and tetrahydrobiopterin, the midpointpotential of the inducible NOS oxygenase heme iron is over 100 mV lower than that of the neuronal NOSoxygenase heme iron. Binding of the substrate alone, cofactor alone, or both combined with the inducibleNOS oxygenase increases the heme iron reduction potential by 112, 52, and 84 mV, respectively. On thebasis of these data, we calculate that the binding affinities of L-arginine and tetrahydrobiopterin increase byabout 80-fold and 8-fold, respectively, for the reduced heme iron form of the enzyme. These data supportinteractive binding of L-arginine and tetrahydrobiopterin in proximity to the inducible NOS heme group, asobserved in the crystal structure of this enzyme. In contrast, addition of L-arginine, tetrahydrobiopterin, orboth to neuronal NOS oxygenase do not markedly change its heme iron midpoint potential, with observedshifts of +19, -18, and -10 mV, respectively. These data explain the contrasting reactivities between thetwo NOS isoforms regarding their different NADPH consumption rates and capacity to support heme ironreduction and are indicative of the regulatory mechanisms that each enzyme employs toward electron transfer.We also examine the effects of three substrate-based inhibitors of NOS on the heme iron midpoint potentials.Among these inhibitors, S-ethylisothiourea decreased the heme potentials of tetrahydrobiopterin-bound inducibleNOS and neuronal NOS by 27 and 24 mV, respectively, N-nitro-L-arginine methyl ester lowered both potentialsbelow -460 mV, and aminoguanidine slightly increased both potentials. This work suggests the following:(1) A thermodynamic block of reductase-catalyzed heme reduction exists in inducible NOS but not in neuronalNOS in the absence of substrate and tetrahydrobiopterin. This reveals distinct heme environments for the twoisoforms. (2) Heme iron reduction thermodynamics in inducible NOS are improved by tetrahydrobiopterinand L-arginine, implying that this isoform is uniquely configured to respond to substrate and pterin control.(3) Some, but not all, inhibitors that reduce electron flux through NOS act by affecting the thermodynamicsof heme iron reduction.