There is increasing evidence that redox reactions at proteins can modify enzyme activities and thus represent a new pathway in the cellular signaling network (reviewed in Refs. 1-5). Molecular oxygen must eventually be involved in such modifications, which can consist of thiol oxidations leading to sulfenic acids or disulfides, sulfoxidation of methionine, nitration of tyrosines, or carbonyl formation (6, 7). thelial cells proved to be sufficient for this oxidative modification (9, 10), although it has been argued that physiological levels of both radicals are not sufficient for tyrosine nitration (11,12). Meanwhile, literature data show that PN generated from superoxide and nitrogen monoxide can nitrate tyrosine (13-15). Moreover, this discrepancy found an explanation in a series of studies proving that the sensitivity and selectivity of heme proteins for tyrosine nitrations reside in an autocatalytic role of the metal ion (16,17). By forming transition metal intermediates from cellular levels of PN, neighboring tyrosines can be selectively attacked without affecting other tyrosines randomly. Following this concept of PN action under physiological conditions, we now extend oxidations by PN also to cysteines. Free and also protein-bound thiols are preferred targets of PN, especially when they are present in their thiolate forms (18 -22). Then a direct two-electron oxidation of the sulfur by the electrophilic PN molecule can take place. It has been reported that the essential ZnCys 2 His center of alcohol dehydrogenase (ADH) can be oxidized by micromolar bolus concentrations of PN (23) and also by ⅐ NO (24) and hypochlorite (25). This can be interpreted as a catalytic action of Zn 2ϩ by converting thiol groups after deprotonation to the more reactive thiolate form. Such zinc thiolate centers may be the subject of a redox regulation by being reversibly oxidized and reduced as part of a signaling mechanism in oxidative stress (26 -28). Therefore, ADH provides a model in which the oxidation of the active site Zn 2ϩ dithiolate center can be studied in the ⅐ NO/O 2 . system by monitoring the enzyme activity. In the present work, we confirm the validity of this model and report on chemically surprising and biologically relevant changes in the oxidizing, nitrating, and nitrosating potential of the system when varying the relative flux rates of ⅐ NO and O 2 . .
EXPERIMENTAL PROCEDURESMaterials-PN was synthesized from ⅐ NO and O 2 . according to the method of Kissner et al. (29). SIN-1 and spermine NONOate were *