Rapid Photochemical Generation of Ubiquinol through a Radical Pathway: An Avenue for Probing Submillisecond Enzyme Kinetics

Abstract

The use of photoreleasable protecting groups ("cages") on bioactive molecules provides a means for the rapid initiation of bimolecular reaction chemistry in biological systems.1,2 In this approach, the protecting group renders an otherwise biologically active molecule inert, so that the molecule can be mixed with an enzyme or other biological target molecule without any reaction taking place. Irradiation of the "caged" molecule leads to the release of the protecting group, so that the biological substrate is free to react with its target biomolecule. Because the bimolecular chemistry can be initiated by a laser pulse, the time frame over which the reaction can be probed is determined by the photochemistry leading to the release of substrate. This time frame can be much shorter than the time scale of milliseconds associated with stopped-flow and other rapid-mixing techniques. For some caged substrates, such as carboxylic acids and phosphates derivatized with benzoin moieties,3-6 release of active substrate is essentially instantaneous upon irradiation. For alcoholic substrates such as quinols, however, release of free substrate has been limited by chemistry that occurs after photocleavage of the cage molecule. When protecting groups such as α-carboxynitrobenzyl7 and 3',5'-bis(carboxymethoxy)benzoin8 are used to derivatize quinols, a carbonate linker is required. Upon irradiation, the quinol is released as a carbonate monoester, and the slow decarboxylation of this species is rate-limiting in generating the free quinol.

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