Trapping methods for probing functional intermediates in nitric oxide synthases and related enzymes

The three mammalian isoforms of nitric oxide synthase (NOS) produce the signalling molecule nitric oxide (NO) from L-arginine, molecular oxygen, and NADPH. NOS-generated NO is essential for many key biochemical processes and aberrant NO production is linked to the pathophysiology of many diseases. Over the past 40 years, the mechanism and structure of NOS have been studied extensively and there are many quality reviews and perspectives documenting the current hypotheses in the field. In this review, as an alternative perspective, we will describe some novel techniques and methodologies that have been used to trap functionally relevant conformational and kinetic states in the catalytic cycles of NOS and some structurally related diflavin oxidoreductase. The methods described in this perspective have enabled characterisation of the complex NOS and diflavin oxidoreductase family members (including cytochrome P450 reductase) and should find future application in other enzyme systems.