Heme insertion, assembly, and activation of apo-neuronal nitric-oxide synthase in vitro.
It has been established that in the case of inducible NO synthase (NOS), a functionally active homodimer is assembled from the heme-deficient monomeric apo-NOS in vitro by the addition of heme, whereas the heme-deficient neuronal isoform (apo-nNOS) is at best only partially activated. In the current study we have discovered that reactive oxygen species, which can be removed by the addition of superoxide dismutase and catalase, destroy the heme and limit the activation of apo-nNOS in vitro. With the use of these improved conditions, we show for the first time that heme insertion is a rapid process that results in formation of a heme-bound monomeric nNOS that is able to form the ferrous-CO P450 complex but is unable to synthesize NO. A slow process requiring more than 90 min is required for dimerization and activation of this P450 intermediate to give an enzyme with a specific activity of approximately 1100 nmol of NO formed/min/mg of protein, similar to that of the native enzyme. Interestingly, the dimer is not SDS-resistant and is not the same dimer that forms in vivo. These studies indicate at least two intermediates in the assembly of nNOS and advance our understanding of the regulation of nNOS.
Bender
A T
AT
Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
Nakatsuka
M
M
Osawa
Y
Y
D000818:Animals; D002374:Catalase / Q000378:metabolism; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006418:Heme / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism
D017382:Reactive Oxygen Species; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D002374:Catalase; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D013482:Superoxide Dismutase