The Redox Status of Bound Pterin Cofactor Determines Whether eNOS Produces NO or Superoxide Anion: [³H]-BH₄ Binding Studies Provide Insights into Vascular Pathophysiology

Abstract

Nitric oxide synthases (NOSs) are a family of bioptero-flavoproteins that convert L-arginine to equimolar quantities of nitric oxide (NO) and L-citrulline. The catalytic mechanism for this reaction is complex, involving electron transfer within NOS from transiently bound NADPH, through flavins in the C-terminal reductase domain, to heme in an N-terminal oxygenase domain — electron transfer is triggered by calmodulin binding to NOS (1). During catalysis, arginine occupies the active site on the oxygenase domain and undergoes two successive heme-dependent monoxygenation reactions, with N^ω-hydroxyarginine formed as an isolatable intermediate (2). Tetrahydrobiopterin (BH₄) is an essential NOS cofactor (3,4), however its precise role in catalysis has not been established. Notably, one molecule of BH₄ is bound in the oxygenase domain of each NOS monomer comprising the functional NOS homodimer. Recent crystal structures have revealed that BH₄ binds tightly at the dimer interface of NOSs and makes multiple hydrogen bonds, including interaction with a heme propionate (5,6).