Laser photoinitiated nitrosylation of 3-electron reduced Nm europaea hydroxylamine oxidoreductase: kinetic and thermodynamic properties of the nitrosylated enzyme.
Hydroxylamine-cytochrome c554 oxidoreductase (HAO) catalyzes the 4-e(-) oxidation of NH(2)OH to NO(2)(-) by cytochrome c554. The electrons are transferred from NH(2)OH 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 microM NO* are photogenerated by laser flash photolysis of N,N'-bis-(carboxymethyl)-N,N'-dinitroso-1,4-phenylenediamine, in a solution containing approximately 1 microM HAO prereduced by 3 e(-)/subunit, the HAO c-heme pool is subsequently oxidized by up to 1 e(-)/HAO subunit. The reaction rate for HAO oxidation shows first-order dependence on [HAO], and zero-order dependence on [NO*] (k(obs) = 1250 +/- 150 s(-)(1)). However, the total HAO oxidized shows hyperbolic dependence on [NO*]. We suggest that NO* first binds reversibly to P(460) giving a {Fe(NO)}(6) moiety. Intramolecular electron transfer (IET) from the c-heme pool then reduces P(460) 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 ferriheme proteins ( approximately 10(4) M(-1)), but much smaller than that for the corresponding ferroheme proteins ( approximately 10(11) M(-1)). The final product generated by nitrosylating 3-e(-) reduced HAO is believed to be the same species obtained by adding NH(2)OH to the fully oxidized enzyme. The experiments described herein suggest that when NH(2)OH and HAO first react, only two of the NH(2)OH electrons end up in the c-heme pool. The other two remain at P(460) as part of an {Fe(NO)}(7) moiety. 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*.
Cabail
Maria Zulema
MZ
Department of Chemistry, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53211, USA.
Kostera
Joshua
J
Pacheco
A Andrew
AA
D002384:Catalysis; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism; D004579:Electron Transport; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism; D043370:Nitrosomonas europaea / Q000201:enzymology*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D013816:Thermodynamics
D003574:Cytochrome c Group; D005296:Ferrous Compounds; D009569:Nitric Oxide; D006418:Heme; D010088:Oxidoreductases; C021401:hydroxylamine dehydrogenase