title: 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)nNOSOX) show four EPR spectra of free radicals. Fully-coupled nNOSOXwith cofactor (tetrahydrobiopterin, BH4) and substrate (l-arginine) forms the typical BH4cation 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 BH4and l-arg is known to produce superoxide. In contrast, we find that nNOSOXwith BH4but 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.
authors.affiliation: Department of Chemistry, The University of Alabama, Tuscaloosa, AL 35487-0336, USA.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Krzyaniak
authors.affiliation: Department of Chemistry, The University of Alabama, Tuscaloosa, AL 35487-0336, USA.
authors.forename: Alex A
authors.initials: AA
authors.lastname: Cruce
authors.affiliation: Department of Chemistry, The University of Alabama, Tuscaloosa, AL 35487-0336, USA.
authors.forename: Preethi
authors.initials: P
authors.lastname: Vennam
authors.affiliation: Department of Chemistry, The University of Alabama, Tuscaloosa, AL 35487-0336, USA.
authors.forename: Molly
authors.initials: M
authors.lastname: Lockart
authors.affiliation: Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center, Houston, TX 77030, USA.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Berka
authors.affiliation: Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center, Houston, TX 77030, USA.
authors.forename: Ah-Lim
authors.initials: AL
authors.lastname: Tsai
authors.affiliation: Department of Chemistry, The University of Alabama, Tuscaloosa, AL 35487-0336, USA. Electronic address: mkbowman@ua.edu.
authors.forename: Michael K
authors.initials: MK
authors.lastname: Bowman
mesh_terms: D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000737:chemistry; D001923:Brain Chemistry; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000737:chemistry; D015870:Gene Expression; D006418:Heme / Q000737:chemistry*; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry* / Q000235:genetics; D010084:Oxidation-Reduction; D000072417:Protein Domains; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics; D012996:Solutions; D013696:Temperature
chemical_list: D005609:Free Radicals; D011994:Recombinant Proteins; D012996:Solutions; D001708:Biopterins; D006418:Heme; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; C003402:sapropterin
keywords: ENDOR; EPR; ESEEM; Nitric oxide synthase; Tetrahydrobiopterin radical