Chemistry of nitric oxide with protein-bound iron sulfur centers. Insights on physiological reactivity
The anaerobic reaction of C. vinosum high-potential iron protein (HiPIP) with nitric oxide has been studied in order to understand the chemical reactivity of NO with protein-bound iron-sulfur clusters. Despite having a solvent inaccessible 4Fe-4S center, native HiPIP reacts with diethylamineNONOate (an NO donor), resulting in protein unfolding and the formation of protein-bound dinitrosyl-iron complexes (DNICs) with a typical g(av) = 2.03 EPR signal. These cysteinyl-coordinated DNICs are directly observed for the first time by use of electrospray ionization-mass spectrometry (ESI-MS) and are found only in a stoichiometry of 2:1 DNIC:protein. Our results suggest that these complexes form only when the protein is folded. ESI-MS also demonstrates that NO-mediated cluster degradation results in nitrosation of the protein at sites other than cysteine. Finally, reactivity comparison of native and Tyr19Leu HiPIP demonstrates solvent accessibility to be an important, but not necessary factor for Fe-S cluster degradation by NO. By use of UV-visible, NMR, and EPR spectroscopies, as well as ESI-MS, we have determined the major products of degradation and elucidate some of the mechanistic issues governing cluster degradation, protein nitrosation, and DNIC formation. Comparisons are made between the nitric oxide chemistry of bacterial HiPIP and other eukaryotic and prokaryotic iron-sulfur proteins that are relevant in vivo targets for NO.
Journal of the American Chemical Society
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