Comparative effects of substrates and pterin cofactor on the heme midpoint potential in inducible and neuronal nitric oxide synthases

Anthony Presta, Anne M Weber-Main, Marian T. Stankovich, Dennis J. Stuehr

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100 Scopus citations

Abstract

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 binding sites of the heme group, as well as of the L-arginine substrate and tetrahydrobiopterin cofactor, are located within the oxygenase domain of the NOS enzymes. Reduction of the heme is the first committed step in catalysis, as this allows for binding and activation of molecular oxygen, followed by oxidative attack on the L-arginine substrate. As with heme groups in other enzymes, the electronic properties of the NOS heme are modified by substrate and cofactor binding in its vicinity. Here we present the first quantitative thermodynamic data of the NOS heme with the determination of the heme midpoint reduction potentials for the neuronal NOS and inducible NOS oxygenase domains. In the absence of L-arginine and tetrahydrobiopterin, the midpoint potential of the inducible NOS oxygenase heme iron is over 100 mV lower than that of the neuronal NOS oxygenase heme iron. Binding of the substrate alone, cofactor alone, or both combined with the inducible NOS oxygenase increases the heme iron reduction potential by 112, 52, and 84 mV, respectively. On the basis of these data, we calculate that the binding affinities of L-arginine and tetrahydrobiopterin increased by about 80-fold and 8-fold, respectively, for the reduced heme iron form of the enzyme. These data support interactive binding of L-arginine and tetrahydrobiopterin in proximity to the inducible NOS heme group, as observed in the crystal structure of this enzyme. In contrast, addition of L-arginine, tetrahydrobiopterin, or both to neuronal NOS oxygenase do not markedly change its heme iron midpoint potential, with observed shifts of +19, -18, and -10 mV, respectively. These data explain the contrasting reactivities between the two NOS isoforms regarding their different NADPH consumption rates and capacity to support heme iron reduction 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 inducible NOS and neuronal NOS by 27 and 24 mV, respectively, N-nitro-L-arginine methyl ester lowered both potentials below - 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 neuronal NOS in the absence of substrate and tetrahydrobiopterin. This reveals distinct heme environments for the two isoforms. (2) Heme iron reduction thermodynamics in inducible NOS are improved by tetrahydrobiopterin and 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 thermodynamics of heme iron reduction.

Original languageEnglish (US)
Pages (from-to)9460-9465
Number of pages6
JournalJournal of the American Chemical Society
Volume120
Issue number37
DOIs
StatePublished - Sep 23 1998

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