Assembly of functional tyrosinase and pMMO metalloenzyme active-site models with biological ligands [electronic resource]

The work in this Doctoral Dissertation is divided between the studies of two copper- and dioxygen-dependent, oxidative enzymes, Tyrosinase (with its active-site homologs Hemocynanin and NspF) and Particulate Methane Monooxygenase (pMMO, the foremost focus of the copper membrane monooxygenase superfamily). The investigation of these enzymes depends on the extremely low temperature, homogenous solution chemistry of synthetic analogs -- low molecular weight models which recapitulate the specific geometric and electronic interactions with copper attributable to coordination of biological ligands within the active sites. By incorporation of the ligand criteria which impart unique function to the enzyme active sites, we can credibly recreate the structural motifs, spectroscopic signatures, and characteristic reactivity of these enzymes for systematized study. First, this work focuses on Tyrosinase, a coupled binuclear protein which activates dioxygen at copper to a dicopper(II) µ-η2:η2-peroxide intermediate. This species is responsible for the electrophilic ortho-hydroxylation of phenolic substrates, namely tyrosine. Subsequent two-electron oxidation of the catechol product to quinone completes the catalytic cycle by reestablishing the dicuprous state. In two lines of investigation, this work explores both the robust self-assembly of the active-site structure from simple components and, separately, the substantiated extension of its catalytic mechanism to a synthetic system compatible with a broad scope of substrates. Examination of the multi-component self-assembly reveals the fundamental stability and innate reactivity of the active site, dictated by chemical forces, and the exploration of the catalytic space suggests that an adapted, biological mechanism is indeed suitable for the manipulation of fine chemicals by the experimental chemist. The second half of this Dissertation probes the unknown chemistry of pMMO, which, at the time of this writing, is reasonably suspected to operate by activation of dioxygen at an asymmetric dicopper site, minimally coordinated by two histidine imidazoles at one copper and chelated by the imidazole and primary amine of an N-terminal histidine at the other. pMMO and the reported homolog Ammonia Monooxygenase, both of the copper membrane monooxygenase superfamily represented in Bacterial and Archaeal domains, catalyze mono-oxygenation of methane and ammonia to methanol and hydroxylamine, respectively, by unestablished mechanisms and intermediates. Because the primary coordination sphere in pMMO seemingly differs substantially from other well-known dicopper active sites, this work leverages structural modeling, specifically incorporating biological histamine ligands, to elucidate the operative dioxygen chemistry and subsequent reactivity, where no previous study credibly proposed substantiated hypotheses. The chief finding of this work is the plausibility of a dicopper(III) bis(µ-oxide) oxygenated intermediate in the catalytic cycle of pMMO and the elevation of biological imidazole and primary amine ligands to strong-field coordination capable of stabilizing the high valent state. The ancillary realization of this investigation is that the unusual, minimal coordination in the pMMO dicopper site could both bias oxygenation to a dicopper(III) complex as well as facilitate reactivity with a recalcitrant substrate by permissible access of methane to the active oxidant. This Dissertation is organized into five Chapters: an introductory account of all work described herein, the study of Tyrosinase self-assembly, followed by the evaluation of synthetic catalysis, and two final Chapters concerning structure, spectroscopy, and reactivity of pMMO through coordination sphere modeling.

Keywords: Chemistry.
Advisor: Stack T. (T. Daniel P.)
Rao Jianghong
Solomon Edward I. advisor. Thesis advisor