Optical Control of enzyme catalysed reactions

Abstract

Industrial synthesis of fine chemicals has recently been shifting from traditional chemical techniques to greener bio based industrial processes. This has happened in tandem with an increase into the understanding of the mechanisms of enzyme catalysis, which has in turn allowed advancements in the area of enzyme engineering. Particular interest in recent years has been paid to both natural photoenzymes as well as using traditionally considered non-photoenzymes to perform photochemistry, as this would allow real time optical control of an enzyme based industrial process. Previous studies have highlighted the old yellow enzyme family (OYE) of flavin based ene-reductase as a potential target for optical non-natural photochemistry. One well characterised member of the OYE is the enzyme pentaerythritol tetranitrate reductase (PETNR). Herein the non-natural photochemistry of PETNR is investigated by a wide range of techniques. Transient visible and infra-red spectroscopy (TRVIS and TRIR) showed that it was possible to photo-induce a temporary single electron transfer from the inactivated nicotinamide cofactors NAD(P)H4, to PETNR bound oxidised flavin. Blue light illuminated-stopped flow spectrometry showed that photo-induced acceleration of the reductive half reaction of PETNR with NADH was possible. Time-dependent density functional theory (TD-DFT) was used to confirm the identity of the transient species seen in the first case and the photochemical mechanism of the second. How IR light can be used to not just investigate the structure of enzymes but also vibrationally control the reaction was also examined. Vibrational control is the ability to control the kinetics of a reaction by excitation of vibrational modes that are coupled to the reaction coordinate. A femtosecond IR laser was used to selectively excite a deuterated C4-H bond stretch to accelerate the reduction of NADH by flavin within PETNR. Further kinetic and computational studies confirmed that this was due to coupling between the vibrational mode of the breaking bond and the reaction coordinate, and not due to heating or the vibration of some other bond. Altogether the results within should help in the development of more novel photoenzymatic reactions, particularly based on ene-reductases. While the vibrational coupling approach will allow direct examination of the reaction coordinate.

Date of Award	1 Aug 2024
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Nigel Scrutton (Supervisor) & Sam Hay (Supervisor)