Creating an artificial functional mimic of the mitochondrial enzyme cytochrome c oxidase (CcO) has been a long-term goal of the scientific community as such a mimic will not only add to our fundamental understanding of how CcO works but may also pave the way for efficient electrocatalysts for oxygen reduction in hydrogen/oxygen fuel cells. Here we develop an electrocatalyst for reducing oxygen to water under ambient conditions. We use site-directed mutants of myoglobin, where both the distal Cu and the redox-active tyrosine residue present in CcO are modelled. In situ Raman spectroscopy shows that this catalyst features very fast electron transfer rates, facile oxygen binding and O–O bond lysis. An electron transfer shunt from the electrode circumvents the slow dissociation of a ferric hydroxide species, which slows down native CcO (bovine 500 s−1), allowing electrocatalytic oxygen reduction rates of 5,000 s−1 for these biosynthetic models.

Catalytic oxidation of organic substrates, using a green oxidant like O 2 , has been a long-term goal of the scientific community. In nature, these oxidations are performed by metalloenzymes that generate highly oxidizing species from O 2 , which, in turn, can oxidize very stable organic substrates, e.g., mono-/dioxygenases. The same oxidants are produced during O 2 reduction/respiration in the mitochondria but are reduced by electron transfer, i.e., reductases. Iron porphyrin mimics of the active site of cytochrome P450 (Cyt P450) are created atop a self-assembled monolayer covered electrode. The rate of electron transfer from the electrode to the iron porphyrin site is attenuated to derive monooxygenase reactivity from these constructs that otherwise show O 2 reductase activity. Catalytic hydroxylation of strong C–H bonds to alcohol and epoxidation of alkenes, using molecular O 2 (with 18 O 2 incorporation), is demonstrated with turnover numbers >10 4 . Uniquely, one of the two iron porphyrin catalysts used shows preferential oxidation of 2° C–H bonds of cycloalkanes to alcohols over 3° C-H bonds without overoxidation to ketones. Mechanistic investigations with labeled substrates indicate that a compound I (Fe IV =O bound to a porphyrin cation radical) analogue, formed during O 2 reduction, is the primary oxidant. The selectivity is determined by the shape of the distal pocket of the catalyst, which, in turn, is determined by the substituents on the periphery of the porphyrin macrocycle.

Myoglobin based biosynthetic models of perturbed cytochrome c oxidase (CcO) active site are reconstituted, in situ, on electrodes where glutamate residues are systematically introduced in the distal site of the heme/Cu active site instead of a tyrosine residue. These biochemical electrodes show efficient 4e–/4H+ reduction with turnover rates and numbers more than 107 M–1 s–1 and 104, respectively. The H2O/D2O isotope effects of these series of crystallographically characterized mutants bearing zero, one, and two glutamate residues near the heme Cu active site of these perturbed CcO mimics are 16, 4, and 2, respectively. In situ SERRS-RDE data indicate complete change in the rate-determining step as proton transfer residues are introduced near the active site. The high selectivity for 4e–/4H+ O2 reduction and systematic variation of KSIE demonstrate the dominant role of proton transfer residues on the isotope effect on rate and rate-determining step of O2 reduction.

Nature has employed heme proteins to execute a diverse set of vital life processes. Years of research have been devoted to understanding the factors which bias these heme enzymes, with all having a heme cofactor, toward distinct catalytic activity. Among them, axial ligation, distal super structure, and substrate binding pockets are few very vividly recognized ones. Detailed mechanistic investigation of these heme enzymes suggested that several of these enzymes, while functionally divergent, use similar intermediates. Furthermore, the formation and decay of these intermediates depend on proton and electron transfer processes in the enzyme active site. Over the past decade, work in this group, using in situ surface enhanced resonance Raman spectroscopy of synthetic and biosynthetic analogues of heme enzymes, a general idea of how proton and electron transfer rates relate to the lifetime of different O 2 derived intermediates has been developed. These findings suggest that the enzymatic activities of all these heme enzymes can be integrated into one general cycle which can be branched out to different catalytic pathways by regulating the lifetime and population of each of these intermediates. This regulation can further be achieved by tuning the electron and proton transfer steps. By strategically populating one of these intermediates during oxygen reduction, one can navigate through different catalytic processes to a desired direction by altering proton and electron transfer steps.

Cytochromes c are small water-soluble proteins that catalyze electron transfer in metabolism and energy conversion processes. Hydrogenobacter thermophilus cytochrome c552 presents a curious case in displaying fluxionality of its heme...