Hydroxylamine-cytochrome c554 oxidoreductase (HAO) catalyzes the 4-e(-) oxidation of NH2OH to NO2- by cytochrome c554. The electrons are transferred from NH2OH to a 5-coordinate heme known as P-460, the active site of HAO. From P-460, c-type hemes transport the electrons through the enzyme to a remote solvent-exposed c-heme, where cyt c554 reduction occurs. When 3-60 muM NO. are photogenerated by laser flash photolysis of N,N-bis-(carboxymethyl)-N,N-dinitroso-1, 4-phenylenediamine, in a solution containing similar to1 muM HAO prereduced by 3 e(-)/subunit, the HAO c-heme pool is subsequently oxidized by up to 1 e(-)/HAO subunit. The reaction rate lot HAO oxidation shows first-order dependence on [HAO], and zero-order dependence on (NO.) (k(abs) = 1250 +/- 150 s(-1)). However, the total HAO oxidized shows hyperbolic dependence on [NO.]. We suggest that NO. first binds reversiblly to P460 giving a {Fe(NO)}(6) moiety. Intramolecular electron transfer (IET) from the c-heme pool then reduces P460 to { Fe(NO)}.(7) The overall binding constant (K) for formation of { Fe(NO)}(7) from free NO. and 3-e(-) reduced HAO was measured at (7.7 +/- 0.6) x10(4) M-1. This value is larger than that for typical ferroheme proteins (similar to10(4) M-1 but much smaller than that for the corresponding ferroheme proteins (similar to10(11) M-1). The final product generated by nitrosylating 3-e(-) reduced HAO is believed to be the same species obtained by adding NH2OH to the fully oxidized enzyme. The experiments described herein suggest that when NH2OH and HAO first react, only two of the NH2OH. electrons end up in the c-heme pool. The other two remain at P460 as part of an {Fe(NO)}(7)(NO)}(7) moiely. These results are discussed in relation to earlier studies that investigated the effect of putting fully oxidized and fully reduced HAO under 1 atm of NO..