Stopped-Flow Infra-Red Spectroscopy of Functioning Nitrogenase

Abstract

Despite extensive biochemical, crystallographic and biomimetic studies, the site and nature of dinitrogen binding to the FeMo cofactor and the chemistry of its subsequent reduction remain unresolved. One reason for this is that only reduced states of the enzyme react with substrates and these can only be generated as mixtures of transient species. Another is the lack of an effective spectroscopic probe of the dinitrogen substrate and its chemistry subsequent to binding to the FeMoCo active site. To address these problems we have developed stopped-flow fourier transform infra-red spectroscopy (SF-FTIR) to probe the dynamics of metalloenzyme-substrate interactions. SF-FTIR is a particularly appropriate probe for nitrogenase chemistry as bound substrates and inhibitors such as N₂, CO, CN⁻, RC=N⁻, and N₃ ⁻ are predicted to exhibit strong infra-red stretching modes within the spectroscopically straightforward “water window” (2600 − 1750 cm⁻¹).