Date

5-2015

Document Type

Dissertation

Degree Name

Ph.D.

Institution

Oregon Health & Science University

Abstract

The overarching goal of our research is to better understand the mechanisms by which metalloproteins obtain and transport their target metal ions. We have characterized the maturation of a copper-containing subunit of cytochrome c oxidase (CuA) in T. thermophilus, as well as the metal ion transport mechanism of a tripartite Cu/Ag ion efflux pump and metallochaperone in E. coli (CusF(CBA)) These characterizations are vital to understanding the basic and applied biochemistry of metal ion homeostasis in living cells, and will inform future research of detrimental mutations and metal ion related diseases in humans as well as possible antimicrobial targets. Cytochrome c oxidase (CcO) is a large terminal respiratory enzyme, belonging to the heme copper oxidase superfamily, and is present in all eukaryotes, and some aerobic prokaryotic organisms. The CuA protein is(~14kD) located in subunit 2 of the ba3–type cytochrome oxidase from Thermus thermophilus, and contains a unique, binuclear Cu center which is able to delocalize a single electron between the two Cu atoms. The assembly of this site within the periplasmic membrane is believed to be mediated by the copper chaperones Sco and/or PCuAC, but the biological mechanisms are still poorly understood. Furthermore, because the CuA center has an electron-delocalized Cu1.5− Cu1.5 center, both Cu(II) and Cu(I) states are implicated in the metallation process. We studied the formation of mixed-valence CuA by stoppedflow UV−vis, EPR, and XAS at both Cu and Se edges, while the formation of fully reduced di-Cu(I) CuA was studied by XAS alone. Our results establish important differences between the metallation reactions of Tt and purple CuA azurin and allow us to extend mechanistic inferences to formation of the di-Cu(I) state which, may be more relevant to biological CuA assembly. We also investigated the periplasmic efflux pump CusCBA and its metallochaperone CusF, which are vital to the detoxification of copper and silver ions in the periplasm of Escherichia coli. We used selenomethionine (SeM) active site labels in a series of biological X-ray absorption studies at the selenium, copper, and silver edges to establish a “switch” role for the membrane fusion protein CusB. We determine that metal-bound CusB is required for activation of cuprous ion transfer from CusF directly to a site in the CusA antiporter, showing for the first time (to our knowledge) the in vitro activation of the Cus efflux pump. This metal-binding site of CusA is unlike that observed in the crystal structures of the CusA protein and is composed of one oxygen and two sulfur ligands. Our results suggest that metal transfer occurs between CusF and apo-CusB, and that, when metal-loaded, CusB plays a role in the regulation of metal ion transfer from CusF to CusA in the periplasm.

Identifier

doi:10.6083/M4T72G5G

Division

Institute of Environmental Health

School

School of Medicine

Available for download on Sunday, June 03, 2018

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