Towards the free energy landscape for catalysis in mammalian nitric oxide synthases

The general requirement for conformational sampling in biological electron transfer reactions catalysed by multi‐domain redox systems has been emphasized in recent years. Crucially, we lack insight into the extent of the conformational space explored and the nature of the energy landscapes associated with these reactions. The nitric oxide synthases ( NOS ) produce the signalling molecule NO through a series of complex electron transfer reactions. There is accumulating evidence that protein domain dynamics and calmodulin binding are implicated in regulating electron flow from NADPH , through the FAD and FMN cofactors, to the haem oxygenase domain, where NO is generated. Simple models based on static crystal structures of the isolated reductase domain have suggested a role for large‐scale motions of the FMN ‐binding domain in shuttling electrons from the reductase domain to the oxygenase domain. However, detailed insight into the higher‐order domain architecture and dynamic structural transitions in NOS enzymes during enzyme turnover is lacking. In this review, we discuss the recent advances made towards mapping the catalytic free energy landscapes of NOS enzymes through integration of both structural techniques (e.g. cryo‐electron microscopy) and biophysical techniques (e.g. pulsed‐electron paramagnetic resonance). The general picture that emerges from these experiments is that NOS enzymes exist in an equilibrium of conformations, comprising a ‘rugged’ or ‘frustrated’ energy landscape, with a key regulatory role for calmodulin in driving vectorial electron transfer by altering the conformational equilibrium. A detailed understanding of these landscapes may provide new opportunities for discovery of isoform‐specific inhibitors that bind at the dynamic interfaces of these multi‐dimensional energy landscapes.