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

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, BH 4 ) and substrate ( l -arginine) forms the typical BH 4 cation radical with an EPR spectrum ~4.0 mT wide and hyperfine tensors similar to reports for a biopterin cation radical in inducible NOS OX (iNOS OX ). With excess thiol, nNOSox lacking BH 4 and l -arg is known to produce superoxide. In contrast, we find that nNOS OX with BH 4 but no l -arg forms two radicals with rather different, fast (~ 250 µs at 5 K) and slower (~ 500 µs at 20 K), electron spin relaxation rates and a combined ~7.0 mT 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 (4.0 mT component) or low-spin (7.0 mT 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.