title: Chloride bound to oxidized cytochrome c oxidase controls the reaction with nitric oxide.
abstract: The reaction of nitric oxide (NO) with oxidized fast cytochrome c oxidase was investigated by stopped-flow, amperometry, and EPR, using the enzyme as prepared or after "pulsing." A rapid reduction of cytochrome a is observed with the pulsed, but not with the enzyme as prepared. The reactive species (lambdamax = 424 nm) reacts with NO at k = 2.2 x 10(5) M-1 s-1 at 20 degreesC and is stable for hours unless Cl- is added, in which case it decays slowly (t1/2 approximately 70 min) to an unreactive state (lambdamax = 423 nm) similar to the enzyme as prepared. Thus, Cl- binding prevents a rapid reaction of NO with the oxidized binuclear center. EPR experiments show no new signals within 15 s after addition of NO to the enzyme as prepared. Amperometric measurements show that the pulsed NO-reactive enzyme reacts with high affinity and a stoichiometry of 1 NO/aa3, whereas the enzyme as prepared reacts to a very small extent (<20%). In both cases, the reactivity is abolished by pre-incubation with cyanide. These experiments suggest that the effect of "pulsing" the enzyme, which leads to enhanced NO reactivity, arises from removing Cl- bound at the oxidized cytochrome a3-CuB site.
authors.affiliation: Department of Biochemical Sciences and Consiglio Nazionale delle Ricerche Center of Molecular Biology, University of Rome "La Sapienza", I-00185 Rome, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Giuffrè
authors.forename: G
authors.initials: G
authors.lastname: Stubauer
authors.forename: M
authors.initials: M
authors.lastname: Brunori
authors.forename: P
authors.initials: P
authors.lastname: Sarti
authors.forename: J
authors.initials: J
authors.lastname: Torres
authors.forename: M T
authors.initials: MT
authors.lastname: Wilson
mesh_terms: D000818:Animals; D002417:Cattle; D002712:Chlorides / Q000378:metabolism*; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000378:metabolism*; D007700:Kinetics; D009569:Nitric Oxide / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding
chemical_list: D002712:Chlorides; D009569:Nitric Oxide; D003576:Electron Transport Complex IV