Dynamics of carbon monoxide binding with neuronal nitric oxide synthase.

The dynamics of CO rebinding with neuronal NO synthase (nNOS) following laser flash photolysis have been investigated from 293 to 77 K in the absence and presence of its substrate L-arginine. The distribution functions of the rate parameters P(k) and of the activation enthalpy P(H) were determined using the maximum entropy method. In a fluid solvent near room temperature, bimolecular rebinding is biphasic, as previously reported by several groups. However, measurement of the rotational correlation time shows that the apparent biphasic rebinding is not relevant to the genuine dynamics of NOS. In addition to native dimeric nNOS, another species (possibly aggregated or partially unfolded conformation) with different hydrodynamic characteristics is responsible for the faster rebinding process. In a rigid environment at low temperature, the geminate internal rebinding is not affected by the presence of the nonnative species. nNOS exhibits a bimodal distribution of CO activation enthalpy with P(H) consisting of two distinct bands with temperature-dependent amplitudes down to 77 K. The similarity of these findings with those recently reported for cytochromes P-450 suggests a common hierarchical organization of conformational substates, with a splitting of each conformational substate into a doublet. Thus, thiolate-coordinated heme proteins are in clear contrast to histidine-coordinated oxygen-transport heme proteins. The present results with nNOS provide additional support to previous arguments incriminating the thiolate ligand as responsible for the splitting of conformational substates.