Document Type


Degree Name



Oregon Health & Science University


The goal of this research is to characterize the contribution of histidine and methionine to function in copper proteins. The emergence of His and Met residues in copper proteins is not new, but the flexible dynamic provided is novel and has been shown to alter function. We have characterized the pH-­‐dependent coordination chemistry involving His-­‐Met residues in cuproproteins peptidylglycine hydroxylating monooxygenase (PHM) and the HM Loop of ATP7A, which is involved in copper translocation. PHM is a member of the unique class of dicopper monooxygenase enzymes that play essential roles in biological processes. Proteins, such as PHM, traverse the secretory pathway where changes in vesicle pH are employed for sorting and post-­‐ translational processing. PHM contains two distinct copper centers, termed CuM and CuH. Despite being separated by an 11 Å solvent filled cavity, the His and Met ligands are essential at each copper center for controlling the catalytic activity. Here, we use spectroscopic techniques such as FTIR and XAS in addition to Michaelis-­‐Menten kinetics of PHM WT and several variants in order to relate copper coordination to activity. PHM WT was found to have optimal catalytic activity at pH 5.8, which is meticulously controlled by the Cu-­‐S(Met) interactions occurring at the catalytic CuM center. An additional Cu-­‐S(Met) interaction was found to bind to the CuH center when the pH decreases below 5 and results in deactivation of the enzyme. Altogether our results demonstrate how one copper center can greatly influence the other’s coordination chemistry, which can largely impact the substrate triggering mechanism. Therefore the coordination of His and Met residues to both centers is critical in controlling the catalytic activity in PHM and may be further regulated by the pH of each individual vesicle. Copper transporter ATPase, ATP7A transports copper from the cytosol into the lumen of vesicles. However, copper release from ATP7A to copper proteins such as PHM, is unknown. Previously, Blackburn and co-­‐workers suggested that copper release involved the HM Loop between TM1 and TM2 on the lumenal side since mutations impacted dephosphorylation, a catalytic step associated with release of the metal ion(s). The transfer of copper is generally mediated by chaperones, but interestingly chaperones do not occur in this system. We have found that factors such as copper loading, redox, and pH contribute to the coordination chemistry and may thus alter function. In addition to the previous HM Loop studies, here we show via XAS spectroscopy that reduced copper is indeed coordinated by a combination of His and Met residues, with a higher degree of Cu-­‐S(Met) coordination at lower pHs. Seleno-­‐methionine labeling for Se K-­‐edge XAS was employed which confirmed and further illustrated the results at the Cu K-­‐edge. Double and triple Met mutants with and without SeM labeling XAS data failed to implicate a specific methionine as critical to the copper coordination, rather they suggested that at least two Met residues acted in concert to control the reactivity. We hypothesize that the His and Met residues in the HM Loop act as a copper chaperone to PHM, both capable of responding to the pH environments encountered.




Institute of Environmental Health


School of Medicine



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