Characterization of Thermal Recovery Activation in Fast Photochromic Flavoprotein Charge-Transfer Complexes

A promising fast, negative, red-absorbing photochromic system based on an intraprotein intermolecular charge transfer (CT) complex between a flavin cofactor and a substrate-analogue inhibitor MXA (methylthioacetate or methylselenoacetate; X = S, Se) within monomeric sarcosine oxidase (MSOX) displays MXA-independent, barrierless, high-yield femtosecond photoactivation associated with CT-interaction disruption coupled to MXA isomerization. Assessing the origin of the known MXA-dependence of the nanosecond thermal recovery requires a very precise determination of activation energy barriers. Here, a newly developed visible pump-probe setup employing two Ti:sapphire femtosecond lasers spanning the picosecond to microsecond time scale was used. Whereas the room temperature thermal recovery time for MSeA is more than double that for MTA (7.5 ns vs 2.9 ns), the dissociation rate, the quantum yield (0.85), and recombination activation enthalpy (21 kJ·mol-1) are MXA-independent. Quantum mechanical calculations rationalize this value as the intrinsic enthalpic barrier for MXA isomerization. The observed variation in thermal recovery is due to the activation entropy, which is negative (on the order of -15 J·mol-1·K-1) and MXA-dependent. This dependence is attributed to a differently ordered transition state. Implications for the design and application of this class of photochromic systems are discussed.