•BH4 radical cation is produced by nNOS with or without substrate.•Spin interactions with heme(III) enhance spin relaxation.•Spin interactions with heme(III) broaden the EPR signal of the BH4 radical.•BH4 plays a decisive role in the tight coupling of reactions in NOS.•Uncoupled NOS reactions can sustain unregulated production of reactive oxygen species.Reaction intermediates trapped during the single-turnover reaction of the neuronal ferrous nitric oxide synthase oxygenase domain (Fe(II)nNOSOX) show four EPR spectra of free radicals. Fully-coupled nNOSOX with cofactor (tetrahydrobiopterin, BH4) and substrate (l-arginine) forms the typical BH4 cation radical with an EPR spectrum ~4.0 mT wide and hyperfine tensors similar to reports for a biopterin cation radical in inducible NOSOX (iNOSOX). With excess thiol, nNOSox lacking BH4 and l-arg is known to produce superoxide. In contrast, we find that nNOSOX with BH4 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.

Electron spin–lattice relaxation of two trityl radicals, d24-OX063 and Finland trityl, were studied under conditions relevant to their use in dissolution dynamic nuclear polarization (DNP). The dependence of relaxation kinetics on temperature up to 100 K and on concentration up to 60 mM was obtained at X- and W-bands (0.35 and 3.5 Tesla, respectively). The relaxation is quite similar at both bands and for both trityl radicals. At concentrations typical for DNP, relaxation is mediated by excitation transfer and spin-diffusion to fast-relaxing centers identified as triads of trityl radicals that spontaneously form in the frozen samples. These centers relax by an Orbach–Aminov mechanism and determine the relaxation, saturation and electron spin dynamics during DNP.