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

• Published in: Free radical biology & medicine 2016-12, Vol.101, p.367-377
• Main Authors: Krzyaniak, Matthew D., Cruce, Alex A., Vennam, Preethi, Lockart, Molly, Berka, Vladimir, Tsai, Ah-Lim, Bowman, Michael K.
• Format: Article
• Language: English
• Subjects: Animals; Biopterins - analogs & derivatives; Biopterins - chemistry; Brain Chemistry; Electron Spin Resonance Spectroscopy; ENDOR; EPR; ESEEM; Free Radicals - chemistry; Gene Expression; Heme - chemistry; Nitric oxide synthase; Nitric Oxide Synthase Type I - chemistry; Nitric Oxide Synthase Type I - genetics; Oxidation-Reduction; Protein Domains; Rats; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Solutions; Temperature; Tetrahydrobiopterin radical
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2016.10.503

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 ~4.0mT 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 (~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. •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.