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We describe a method for addressing redox enzymes adsorbed on a carbon electrode using synchrotron infrared microspectroscopy combined with protein film electrochemistry. Redox enzymes have high turnover frequencies, typically 10-1000 s-1, and therefore, fast experimental triggers are needed in order to study subturnover kinetics and identify the involvement of transient species important to their catalytic mechanism. In an electrochemical experiment, this equates to the use of microelectrodes to lower the electrochemical cell constant and enable changes in potential to be applied very rapidly. We use a biological cofactor, flavin mononucleotide, to demonstrate the power of synchrotron infrared microspectroscopy relative to conventional infrared methods and show that vibrational spectra with good signal-to-noise ratios can be collected for adsorbed species with low surface coverages on microelectrodes with a geometric area of 25 ? 25 μm². We then demonstrate the applicability of synchrotron infrared microspectroscopy to adsorbed proteins by reporting potential-induced changes in the flavin mononucleotide active site of a flavoenzyme. The method we describe will allow time-resolved spectroscopic studies of chemical and structural changes at redox sites within a variety of proteins under precise electrochemical control. [ABSTRACT FROM AUTHOR]

Deuterium-labeled nicotinamide cofactors such as [4- 2 H]-NADH can be used as mechanistic probes in biological redox processes and offer a route to the synthesis of selectively [ 2 H] labeled chemicals via biocatalytic reductive deuteration. Atom-efficient routes to the formation and recycling of [4- 2 H]-NADH are therefore highly desirable but require careful design in order to alleviate the requirement for [ 2 H]-labeled reducing agents. In this work, we explore a suite of electrode or hydrogen gas driven catalyst systems for the generation of [4- 2 H]-NADH and consider their use for driving reductive deuteration reactions. Catalysts are evaluated for their chemoselectivity, stereoselectivity, and isotopic selectivity, and it is shown that inclusion of an electronically coupled NAD + -reducing enzyme delivers considerable advantages over purely metal based systems, yielding exclusively [4 S - 2 H]-NADH. We further demonstrate the applicability of these types of [4 S - 2 H]-NADH recycling systems for driving reductive deuteration reactions, regardless of the facioselectivity of the coupled enzyme., Competing Interests: The authors declare the following competing financial interest(s): A patent application detailing some of this research was filed through Oxford University Innovation (Feb 2018)., (© 2021 American Chemical Society.)