Abstract
In enzyme systems where fast motions are thought to contribute to H-transfer efficiency, the distance between hydrogen donor and acceptor is a very important factor. Sub-ångstrom changes in donor-acceptor distance can have a large effect on the rate of reaction, so a sensitive probe of these changes is a vital tool in our understanding of enzyme function. In this study we use ultrafast transient absorption spectroscopy to investigate the photoinduced electron transfer rates, which are also very sensitive to small changes in distance, between coenzyme analog, NAD(P)H4, and the isoalloxazine center in the model flavoenzymes morphinone reductase (wild-type and selected variants) and pentaerythritol tetranitrate reductase (wild-type). It is shown that upon addition of coenzyme to the protein the rate of photoinduced electron transfer is increased. By comparing the magnitude of this increase with existing values for NAD(P)H4-FMN distances, based on charge-transfer complex absorbance and experimental kinetic isotope effect reaction data, we show that this method can be used as a sensitive probe of donor-acceptor distance in a range of enzyme systems.