Abstracts

In the early 1970s, Giuseppe Cilento (IQUSP), Emil White (Johns Hopkins University), and Angelo Lamola (AT&T Bell Laboratories) postulated that typically photochemical reactions could occur in vivo in the absence of light. This paradoxical hypothesis named "photochemistry in the dark" was chemically anchored by the synthesis and study of 1,2-dioxetanes and 1,2-dioxetanones, whose thermolysis generates excited carbonyl species in the triplet excited state, which has relatively long lifetimes and emit ultra-weak chemiluminescence. The oxidation of isobutanal (IBAL) catalyzed by horseradish peroxidase (HRP) is a model study of an enzymatic reaction that generates triplet acetone via a high-energy intermediate derived from a hypothetical 1,2-dioxetane. The generation of triplet carbonyls through dioxetane intermediates by ferrylmyoglobin (ferrylMb) has been reported for acetoacetate and methylacetoacetate substrates, yielding, respectively, methylglyoxal and biacetyl in the triplet excited state. Here, we aim to investigate the role of ferrylMb as a peroxidase, generating triplet species, through a comparative study with the already known activity of HRP. The HRP/H₂O₂/IBAL and Mb/H₂O₂/IBAL systems were characterized through chemiluminescence studies by varying the concentration of components and pH. Oxygen consumption experiments were conducted to associate the proposed O₂-dependent mechanism with the observed chemiluminescence. The HRP system reveals a zero-order kinetics on O₂ and a kinetic constant dependent on IBAL concentration similarly to the ultra-weak light emission. The Mb reaction shows roughly a first-order kinetics on O₂, as already reported for other peroxidase/substrate systems. Both systems were challenged with sorbic acid, a well-known triplet species quencher, and L-Tyr, which can act as a quencher. Applying the classic Stern-Volmer treatment, the kinetic quenching constants found for sorbic acid from HRP and Mb systems were, respectively, 8.02 x 10⁸ M^-1s^-1 and 1.56 x 10⁹ M^-1s^-1, considering τ = 1.2 μs for triplet acetone lifetime in aqueous solution. Both data are consistent with a collisional quenching process (diffusional coefficient in water ~ 5 x 10⁹ M^-1s^-1). The two times difference between the values can be attributed to structural differences between the two proteins, such as water-exposure active sites. Importantly, a quadratic function was observed for the L-Tyr Stern-Volmer plots, a typical behavior of concomitant dynamic and static processes. Thus, the quenching mechanism may be granted to the formation of L-Tyr-IBAL complex into the active site of enzyme, not observed for sorbic acid. Altogether, these data suggest that myoglobin seems to behave as a peroxidase on IBAL oxidation similarly to HRP, which can be harnessed to elucidate potential involvement of myoglobin and excited triplet species in carbonyl stress-related biological processes.