Dynamics of nitric oxide in the active site of reduced cytochrome c oxidase aa(3)

Nitric oxide (NO) is involved in the regulation of respiration by acting as a competitive ligand for molecular oxygen at the binuclear active site of cytochrome c oxidase. The dynamics of NO in and near this site are not well understood. We performed flash photolysis studies of NO from heme a(3) in cytochrome c oxidase from Paracoccus denitrificans, using femtosecond trans ient absorption spectroscopy. The formation of the product state-the unliga nded heme a3 ground state-occurs in a similar stepwise manner (period simil ar to 700 fs) as previously observed for carbon monoxide photolysis from th is enzyme and interpreted in terms of ballistic ligand motions in the activ e site on the subpicosecond time scale [Liebl, U., Lipowski, G., Negrerie, M., Lambry, J.-C., Martin, J.-L., and Vos, M. H. (1999) Nature 401, 181-184 ]. A fraction (similar to 35% at very low NO concentrations) of the dissoci ated NO recombines with heme a(3) in 200-300 ps. The presence of this recom bination phase indicates that a transient bond to the second ligand-binding site, a copper atom (CuB), has a short lifetime or may not be formed. Incr easing the NO concentration increases the recombination yield on the hundre ds of picoseconds time scale. This effect, unprecedented for heme proteins, implies that, apart from the one NO molecule bound to heme a3, a second NO molecule can be accommodated in the active site, even at relatively low (s ubmicromolar) concentrations. Models for NO accommodation in the active sit e, based on molecular dynamics energy minimizations are presented. Pathways for NO motion and their relevance for the regulation of respiration are di scussed.