Dinitrosyliron complexes and the mechanism(s) of cellular protein nitrosothiol formation from nitric oxide

Nitrosothiols (RSNO), formed from thiols and metabolites of nitric oxide (center dot NO), have been implicated in a diverse set of physiological and pathophysiological processes, although the exact mechanisms by which they are formed biologically are unknown. Several candidate nitrosative pathways involve the reaction of center dot NO with O-2, reactive oxygen species (ROS), and transition metals. We developed a strategy using extracellular ferrocyanide to determine that under our conditions intracellular protein RSNO formation occurs from reaction of center dot NO inside the cell, as opposed to cellular entry of nitrosative reactants from the extracellular compartment. Using this method we found that in RAW 264.7 cells RSNO formation occurs only at very low (< 8 mu M) O-2 concentrations and exhibits zero-order dependence on center dot NO concentration. Indeed, RSNO formation is not inhibited even at O-2 levels < 1 mu M. Additionally, chelation of intracellular chelatable iron pool (CIP) reduces RSNO formation by > 50%. One possible metal-dependent, O-2-independent nitrosative pathway is the reaction of thiols with dinitrosyliron complexes (DNIC), which are formed in cells from the reaction of center dot NO with the CIP. Under our conditions, DNIC formation, like RSNO formation, is inhibited by approximate to 50% after chelation of labile iron. Both DNIC and RSNO are also increased during overproduction of ROS by the redox cycler 5,8-dimethoxy-1,4-naphthoquinone. Taken together, these data strongly suggest that cellular RSNO are formed from free center dot NO via transnitrosation from DNIC derived from the CIP. We have examined in detail the kinetics and mechanism of RSNO formation inside cells.