Investigating the diverse role of metals in protein structure and function

Metal ions are essential to life where they play a critical role in over a third of all proteins. The ability of metals to gain and lose electrons enables a variety of functions ranging from electron transport and reaction catalysis to structural stabilization. This thesis explores how exogenous and native metal ions interact with proteins to disrupt structure or to provide structural stability. This work examines: 1) how a synthetic copper protease disrupts protein structure during cleavage; 2) how two heme-containing proteins are regulated allosterically; 3) the identity of the Fe(III) cysteine(thiolate) ligand in the regulator of CO metabolism (RcoM) from Burkholderia xenovorans; and 4) the insertion of CoPPIX into human cystathionine beta-synthase (hCBS).;Utilization of a variety of spectroscopic techniques revealed that incubating bovine serum albumin (BSA), aldolase and glyceraldehyde-3-phopshate dehydrogenase (GAPDH) with [Cu(1,4,7-triazocyclononane)]Cl2 results in cleavage and protein aggregation via disruption of tertiary structures, but does not induce fibril formation. In-gel trypsin digestion coupled with MALDI-MS was used to identify the sites of aldolase cleavage. The copper complex accessed buried residues during cleavage.;Mass spectrometry enabled studies on two allosterically regulated proteins, the CO-sensing carbon monoxide oxidation activator (CooA) protein from Rhodospirillum rubrum and CBS from Drosophila melanogaster (DmCBS). Initial hydrogen/deuterium exchange MS experiments with CooA confirmed that the DNA-binding and the heme-binding domains are independently folded. Mass spectrometry revealed that DmCBS does not bind the allosteric regulator AdoMet, even under saturating conditions, unlike AdoMet-regulated hCBS.;The BxRcoM protein is a CO-responsive transcription factor that binds heme with an Fe(III) Cys/His ligation motif. Expression and purification of variants at all three cysteines in BxRcoM, C94S, C127S and C130S, allowed spectroscopic identification of Cys 94 is as the native cysteine(thiolate) ligand to the Fe(III) heme in BxRcoM.;CBS is a PLP-dependent enzyme bearing a heme cofactor believed to be involved in structural stabilization. Metal analysis and spectroscopic characterization of CoPPIX-substituted human CBS (CoCBS) revealed that CoCBS contained 92% CoPPIX and retained the native His/Cys metal coordination. CoCBS was fully active. Together these results support a structural role for heme in hCBS.