Conformational and thermodynamic control of electron transfer in neuronal nitric oxide synthase.

Multiple solution-state techniques have been employed in investigating the nature and control of electron transfer in the context of the proposed "domain shuffle hypothesis" for intraprotein electron transfer inferred from the crystal structure of the nitric oxide synthase reductase domain. NADPH analogues and fragments have been used to map those regions of this substrate that are important in eliciting a conformational change, observed in both the fluorescence emission of the flavin cofactors of the enzyme and the EPR spectra of the FMN flavosemiquinone state. EPR and UV-visible potentiometric methods have demonstrated a substantial calmodulin-dependent perturbation in the midpoint reduction potentials of the redox couples of both flavin cofactors, in contrast to a previous report [Noble, M. A., et al. (1999) Biochemistry 38, 16413-16418]. These studies support a model in which FMN domain mobility, triggered by Ca2+-calmodulin binding and antagonized by substrate binding, facilitates electron transfer in nitric oxide synthase through conformational change and effects a major change in the midpoint reduction potentials of the flavin redox couples. These results are discussed in light of the recent crystal structure of the NADPH-locked reductase domain.