Spectroscopic and computational investigation of adenosylcobalamin-dependent enzymes and a membrane-bound fatty acid desaturase

Coenzyme B12 (5'-deoxyadenosylcobalamin, AdoCbl) is a remarkable cofactor derived from Vitamin B12 and a rare example of a natural, biologically-relevant organometallic compound. All mechanisms employed by AdoCbl-dependent enzymes are initiated by homolysis of the cofactor's Co--C bond to form cob(II)alamin and a highly reactive adenosyl radical. To investigate the interactions between cofactor, enzyme, and substrate that lead to activation of the Co--C bond, spectroscopic and computational techniques were applied to free and enzyme-bound AdoCbl, cob(II)alamin, and related species. This research highlighted the mechanistic implications of differences between AdoCbl-dependent isomerases and eliminases, in which the former bind AdoCbl by substituting its lower axial ligand with a histidine residue.;Spectroscopic changes caused by reducing the electron-donating character of the lower axial ligand of cofactor analogues were found to be similar to those observed when cob(II)alamin binds to isomerases. This observation was used to suggest that the basicity of the coordinating histidine is modulated during turnover by proton uptake into a conserved hydrogen bonding network, thus preferentially stabilizing the cob(II)alamin state. In contrast, an experimentally-validated computational model of AdoCbl bound to the eliminase ethanolamine ammonia lyase (EAL) revealed that destabilization of AdoCbl upon the binding of substrate triggers Co--C bond homolysis. A subsequent computational study confirmed the importance of two active-site residues, N193alpha and Y241beta, and proposed future AdoCbl analogues and EAL variants for spectroscopic characterization.;Stearoyl-CoA desaturase (SCD) is a membrane-bound, non-heme diiron enzyme (NHFe2) that converts stearoyl-CoA to oleyl-CoA alongside the reduction of O2 to two water molecules. The active-site architecture of SCD is novel among NHFe2 enzymes and includes an Fe--Fe separation that precludes the possibility of activating O2 by binding it as a bridging ligand. A computational investigation of SCD allowed for proposal of a likely O2 binding site and a putative reaction mechanism.;Lastly, an interactive program that facilitates the implementation of computational analyses of whole-protein systems was written. This program, which has been used by several graduate and postdoctoral researchers and will soon be used by undergraduate researchers, helps lower barriers to the theoretical study of biomolecules.