Kelath Murali Manoj

Chloroperoxidase, a janus enzyme.

Chloroperoxidase is a versatile fungal heme-thiolate protein that catalyzes a variety of one-electron and two-electron oxidations. We report here that the alkylation of an essential histidine residue showed no effect on the one-electron peroxidations but inhibited two-electron oxidations. The pH profiles of different peroxidative substrates showed optimal activities at varying pH values for the same enzyme. 2-Allylphenol and substituted ortho-phenolics showed efficient peroxidations. Also, substrates excluded from the active site (or with no favorable positioning at the heme center or heme edge) were converted in the peroxidation reaction. While hydrogen peroxide serves as the superior activator in the two-electron oxidations, small alkylhydroperoxides give much better rates for peroxidation reactions. All the above observations indicate that one-electron oxidations are mechanistically quite different from the two-electron oxidations catalyzed by chloroperoxidase. We propose that the peroxidatic substrates interact predominantly outside the heme active site, presumably at the surface of the enzyme.

Utilization of peroxide and its relevance in oxygen insertion reactions catalyzed by chloroperoxidase.

Chloroperoxidase (CPO) catalyzed oxygen insertions are highly enantioselective and hence of immense biotechnological potential. A peroxide activation step is required to give rise to the compound I species that catalyzes this chiral reaction. A side reaction, a catalase type peroxide dismutation, is another feature of CPOs versatility. This work systematically investigates the utilization of different peroxides for the two reactions, i.e. the catalase type reaction and the oxygen insertion reaction. For the oxygen insertion reaction, indene and phenylethyl sulfide were chosen as substrate models for epoxidation and sulfoxidation respectively. The results clearly show that CPO is stable towards hydrogen peroxide and has a total number of turnovers near one million prior to deactivation. The epoxidation reactions terminate before completion because the enzyme functioning in its catalatic mode quickly removes all of the hydrogen peroxide from the reaction mixture. Sulfoxidation reactions are much faster than epoxidation reactions and thus are better able to compete with the catalase reaction for hydrogen peroxide utilization. A preliminary study towards optimizing the reaction system components for a laboratory scale synthetic epoxidation is reported.

Epoxidation of indene by chloroperoxidase

The stereochemistry of the chloroperoxidase-catalyzed epoxidation of indene has been elucidated. In aqueous solution the intial epoxide product is not stable and opens to form the cis-trans diols. When the reaction was carried out in the absence of water, the epoxide enantiomers could be isolated. Under these conditions in 1R2S enantiomer was formed in approximately 30% ee.

Electron transfer amongst flavo- and hemo-proteins: diffusible species effect the relay processes, not protein–protein binding

Hitherto, electron transfer (ET) between redox proteins has been deemed to occur via donor–acceptor binding, and diffusible reactive species are considered as deleterious side-products in such systems. Herein, ET from cytochrome P450 reductase (CPR, an animal membrane flavoprotein) and horseradish peroxidase (HRP, a plant hemoprotein) to cytochrome c (Cyt c, a soluble animal hemoprotein) was probed under diverse conditions, using standard assays. ET in the CPR-Cyt c system was critically inhibited by cyanide and sub-equivalent levels of polar one-electron cyclers like copper ions, vitamin C/Trolox and superoxide dismutase. In the presence of lipids, inhibition was also afforded by amphipathic molecules vitamin E, palmitoyl-vitamin C and the membrane hemoprotein, cytochrome b5. Such non-specific inhibition (by diverse agents in both aqueous and lipid phases) indicated that electron transfer/relay was effected by small diffusible agents, whose lifetimes are shortened by the diverse radical scavengers. When CPR was retained in a dialysis membrane and Cyt c presented outside in free solution, ET was still observed. Further, HRP (taken at nM levels) catalyzed oxidation of a phenolic substrate was significantly inhibited upon the incorporation of sub-nM levels of Cyt c. The findings imply that CPR-Cyt c or HRP-Cyt c binding is not crucial for ET. Further, fundamental quantitative arguments (based on diffusion/collision) challenge the erstwhile protein–protein binding-assisted ET hypothesis. It is proven beyond reasonable doubt that mobile and diffusible electron carriers (ions and radicals) serve as “redox-relay agents” in the biological ET models/setup studied.

Murburn Concept: A Molecular Explanation for Hormetic and Idiosyncratic Dose Responses

Recently, electron transfers and catalyses in a bevy of redox reactions mediated by hemeproteins were explained by murburn concept. The term “murburn” is abstracted from “ mur ed burn ing” or “ m ild u n r estricted burn ing” and connotes a novel “ m olecule- u nbound ion– r adical” interaction paradigm. Quite unlike the genetic regulations and protein-level affinity-based controls that govern order and specificity/selectivity in conventional treatments, murburn concept is based on stochastic/thermodynamic regulatory principles. The novel insight necessitates a “reactivity outside the active-site” perspective, because select redox enzymatic activity is obligatorily mediated via diffusible radical/species. Herein, reactions employing key hemeproteins (as exemplified by CYP2E1) establish direct experimental connection between “additive-influenced redox catalysis” and “unusual dose responses” in reductionist and physiological milieu. Thus, direct and conclusive molecular-level experimental evidence is presented, supporting the mechanistic relevance of murburn concept in “maverick” concentration-based effects brought about by additives. Therefore, murburn concept could potentially explain several physiological hormetic and idiosyncratic dose responses.