The tetrahydrobiopterin radical interacting with high- and low-spin heme in neuronal nitric oxide synthase - A new indicator of the extent of NOS coupling

Abstract

Reaction intermediates trapped during the single-turnover reaction of the neuronal ferrous nitric oxide synthase oxygenase domain (Fe(II)nNOS_OX) show four EPR spectra of free radicals. Fully-coupled nNOS_OX with cofactor (tetrahydrobiopterin, BH₄) and substrate (l-arginine) forms the typical BH₄ cation radical with an EPR spectrum ~4.0mT wide and hyperfine tensors similar to reports for a biopterin cation radical in inducible NOS_OX (iNOS_OX). With excess thiol, nNOSox lacking BH₄ and l-arg is known to produce superoxide. In contrast, we find that nNOS_OX with BH₄ but no l-arg forms two radicals with rather different, fast (~250μs at 5K) and slower (~500μs at 20K), electron spin relaxation rates and a combined ~7.0mT wide EPR spectrum. Rapid freeze-quench CW- and pulsed-EPR measurements are used to identify these radicals and their origin. These two species are the same radical with identical nuclear hyperfine couplings, but with spin-spin couplings to high-spin (4.0mT component) or low-spin (7.0mT component) Fe(III) heme. Uncoupled reactions of nNOS leave the enzyme in states that can be chemically reduced to sustain unregulated production of NO and reactive oxygen species in ischemia-reperfusion injury. The broad EPR signal is a convenient indicator of uncoupled nNOS reactions producing low-spin Fe(III) heme.

Keywords: ENDOR; EPR; ESEEM; Nitric oxide synthase; Tetrahydrobiopterin radical.