Dynamics of Nitric Oxide in the Active Site of Reduced Cytochrome c Oxidase aa₃

Abstract

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 a3 in cytochrome c oxidase from Paracoccus denitrificans, using femtosecond transient absorption spectroscopy. The formation of the product state--the unliganded heme a3 ground state--occurs in a similar stepwise manner (period approximately 700 fs) as previously observed for carbon monoxide photolysis from this enzyme and interpreted in terms of ballistic ligand motions in the active site on the subpicosecond time scale [Liebl, U., Lipowski, G., Négrerie, M., Lambry, J.-C., Martin, J.-L., and Vos, M. H. (1999) Nature 401, 181-184]. A fraction (approximately 35% at very low NO concentrations) of the dissociated NO recombines with heme a3 in 200-300 ps. The presence of this recombination 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. Increasing the NO concentration increases the recombination yield on the hundreds 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 (submicromolar) concentrations. Models for NO accommodation in the active site, based on molecular dynamics energy minimizations are presented. Pathways for NO motion and their relevance for the regulation of respiration are discussed.

NO Binding and Dynamics in Reduced Heme−Copper Oxidases aa3 from Paracoccus denitrificans and ba3 from Thermus thermophilus

“…This behavior, which contrasts with that of the aa 3 enzyme (see the Materials and Methods and ref 13), is consistent with the proposed high affinity for NO of heme a 3 in CcO ba 3 (10). It also implies that under these conditions NO is not consumed by NOreductase activity of the enzyme and that all active sites accommodate 1 NO molecule.…”

“…Figure 7 shows relevant features of them. As reported in our previous modeling (13), the models show substantial motion of CuB upon its binding to NO, with weakening of the CuBHis 276 interaction. In addition, the present representation makes clear that the heme-bound NO rotates substantially; the Fe-N-O plane is nearly parallel to that of His 411 (14°) in the 1-NO model but nearly perpendicular (77°) in the 2-NO model.…”

“…The earlier observed effects of NO/enzyme stoichiometry on the picosecond heme-NOrebinding kinetics in aa 3 (13) were now extended to and also observed on longer time scales. Along with the novel EPR characterization and the comparison with ba 3 , they further confirm the assessment that two NO molecules can be accommodated by the active site at relatively low NO concentrations and allow proposing structural characteristics of the NO accommodation.…”

“…Molecular modeling of the NO-bound structures of the active site of CcO aa 3 from P. denitrificans was performed starting from previously generated energy-minimized structures (13). To avoid trapping of these structures in local minima, molecular dynamics simulations with an integration time of 1 fs were performed on these structures and the temperature was raised to 300 K in steps of 10 K, with an equilibration time of 1 ps at each temperature.…”

“…Using this property, we have reported a pronounced effect of the NO/ enzyme stoichiometry on the picosecond NO-rebinding kinetics after dissociation from reduced CcO aa 3 from Paracoccus denitrificans (13), an effect which we ascribed to the presence of two NO molecules in the active site at higher NO concentrations. One NO molecule binds to heme a 3 , but the second NO binding site was not clearly identified.…”