(Bio)chemistry of Coenzyme B12
Coenzyme B12, or adenosylcobalamin (AdoCbl), is a complex cofactor containing an organometallic Co–C bond. This Co–C bond lies at the heart of AdoCbl reactivity and, in the presence of enzyme and substrate, cleaves homolytically to produce a primary carbon radical and a Co(II) radical species. Our lab studies the structural and electronic factors that promote Co–C homolysis in different classes of AdoCbl-dependent enzymes. We have found that these different classes of AdoCbl-dependent enzymes, which employ unique AdoCbl binding modes, utilize distinct mechanisms for Co–C activation but still achieve a trillion-fold rate acceleration for Co–C homolysis.
Thiol Dioxygenases (TDOs)
Cysteine dioxygenase (CDO) is a mono-nuclear non-heme Fe(II)-dependent enzyme that catalyzes the oxidation of cysteine to cysteine sulfinic acid with high specificity. CDO features a 3-histidine ligation sphere and a unique covalent Cys-Tyr crosslink. Our lab investigates the function of these unusual features in eukaryotic and bacterial CDOs and their role in the catalytic mechanism.
Cysteamine dioxygenase (ADO) is a related enzyme that catalyzes the oxidation of cysteamine. ADO has additionally shown activity with N-terminal cysteine residues. Our lab explores the substrate specificity and redox gating of ADO.
Spectroscopy
Our lab employs several spectroscopic techniques to investigate the metalloprotein active site in various enzymatic states. These include electronic absorption, magnetic circular dichroism (MCD), resonance Raman (rR), and electron paramagnetic resonance (EPR) spectroscopies, which are sensitive to different metal ion oxidation states and substrate coordination. The information from these techniques provide insight into the geometric and electronic properties of the active site.
Computations
Our lab generate quantum mechanics/molecular mechanics (QM/MM) optimizations of whole proteins, using QM for the active site and MM for the rest of the protein. Our computational models are validated via time dependent density functional theory (TD-DFT), single point DFT calculations, frequency calculations, and EPR simulations. We use these computational models to assess the geometric and electronic structures of reaction intermediates that are not readily studied experimentally.
Biochemistry
Our students working with TDOs express and purify proteins and characterize them with biochemical techniques including gel electrophoresis, mass spectrometry, and X-ray crystallography. We have adapted several kinetic assays to assess the activity of enzyme variants with different substrates. We are currently working on site-specific incorporation of unnatural amino acids.
