Project Summary/Abstract.
The interactions of coupled binuclear copper sites and dioxygen have been extensively studied in contexts crucial
to life. This contrasts with analogous interactions between mononuclear copper sites and dioxygen, which are less
well understood despite their importance in clinical, biological, and industrial settings. As such, furthering our
understanding of these enzymes' mechanisms of action would have far reaching implications across a wide set of
disciplines. This project focuses on mechanistic elucidation of the oxidative behaviors of three different monooxygenases featuring single copper active sites: tyramine ß-monooxygenase (TßM), lytic polysaccharide monooxygenase
(LPMO), and particulate methane monooxygenase (pMMO). The first, TßM, is closely related to human dopamine
ß-monooxygenase (DßM) and participates in invertebrate neurotransmitter regulation. As a noncoupled binuclear
copper enzyme, it features two copper centers separated by 11 Å, only one of which engages with O2. Outstanding questions on the timing of O2 binding to the reduced enzyme, hydrogen atom abstraction (HAA), and
intermetallic electron transfer persist. The second enzyme, LPMO, has large industrial applications in renewable
biofuels and has also been implicated as a virulence factor in several pathogens. Its active site comprises a single
copper ion coordinated to two histidine residues and the N-terminal amine in a rare "histidine brace" geometry.
Computational results inform the planned experiments, suggesting several mechanistic possibilities; though, some
recent reports have questioned the role of O2 in favor of H2O2. The last enzyme, pMMO, is important in conversion of methane, a dangerous greenhouse gas, into methanol, a renewable fuel. It has long been thought to
possess a coupled binuclear copper active site, but has recently been reappraised to have a mononuclear copper site
also engaged in a histidine brace structural motif. This new suggestion means there is little mechanistic insight
available, though it draws parallels between pMMO and LPMO. The project ultimately aims to shed light on
how these enzymes oxidize their substrates, and to uncover useful and generalizable structure-function relations
to be exploited in further clinical and industrial applications. Our specific aims involve investigation of each class
of enzyme using a battery of spectroscopies to uncover informative intermediates, capitalizing on the extensive
instrumentation and experience available in the Solomon lab. As many of these transformations involve paramagnetic species, electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) experiments
will allow direct interrogation of the copper center, particularly in combination with rapid freeze quench (RFQ)
techniques. Additionally, stopped ow absorption, resonance Raman (rR), X-ray absorption (XAS), and X-ray
emission (XES) will be heavily employed, especially when studying diamagnetic states. All of these studies will be
supported by thorough computational investigations using density functional theory (DFT) methods, which will
allow for further insight into the electronic structures and reaction energies. The training plan involves immersion
in these spectroscopic techniques and in the field of bioinorganic chemistry, all of which are new to the applicant.