Metalloproteins involved in the assembly of cytochrome c oxidase: in vitro and in vivo consequences of metal binding and oligomerization

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Publication Type dissertation
School or College School of Medicine
Department Biochemistry
Author Coyne, Harold Jerome
Title Metalloproteins involved in the assembly of cytochrome c oxidase: in vitro and in vivo consequences of metal binding and oligomerization
Date 2007-05
Description All forms of cytochrome c oxidase (CcO) contain the three essential core subunits. Eukaryotic organisms contain additional peripheral subunits in their CcO compared to bacterial enzymes. The roles of the peripheral subunits have not been completely addressed, but they appear to have subtle regulatory and structural purposes. Bovine CoxVb is a peripheral subunit of CcO that binds zinc. Yeast Cox4 is the homolog of CoxVb. Cox4 is necessary for the assembly of yeast CcO. Cox4 has only three of the four zinc-binding cysteine residues conserved with CoxVb, and no in vitro or in vivo evidence has been presented concerning zinc binding by the yeast subunit. We used NMR, UV/VIS spectroscopy, and EXAFS measurements to structurally characterize Cox4. Three cysteine residues and a histidine residue from Cox4 are involved in zinc coordination. Using biochemical assays we show the significance of zinc binding by Cox4 for CcO assembly. Three copper ions are found in each monomer of CcO, and the ions are important for catalysis. It is known that chaperones are important for the coordinated delivery of copper to CcO. Two of these proteins, Cox 17 and Cox 19, coordinate copper in vivo, and both are necessary for CcO assembly. We conducted structural studies on both proteins to identify the physiologically relevant isoform of Cox 17, and to determine if Coxl9 has a conserved fold similar to other intermembrane space proteins. We took a mutagenesis approach to identify the physiological relevant form of Coxl7, by creating a mutant that would favor one oligomeric state over the others. The mutants were examined in vivo by transformation in a Acoxi 7 yeast strain and in vitro by gel filtration of recombinant expressed protein. I found that purification of recombinant Coxl7 was sensitive to the lysis conditions. Consequently, the purified Coxl7 may not be reflective of the in vivo oligomer state. Finally, our NMR analysis determined that Coxl9 and Coxl7, both intermembrane space proteins, are not structurally similar. Site directed mutagenesis was applied to examine residue differences between Cox 17 and Coxl9. We found that the residues investigated are potentially involved in promoting higher order oligomerization states.
Type Text
Publisher University of Utah
Subject Cytochrome oxidase
Subject MESH Metalloproteins; Cytochromes
Dissertation Institution University of Utah
Dissertation Name PhD
Language eng
Relation is Version of Digital reproduction of "Metalloproteins involved in the assembly of cytochrome c oxidase: in vitro and in vivo consequences of metal binding and oligomerization." Spencer S. Eccles Health Sciences Library. Print version of "Metalloproteins involved in the assembly of cytochrome c oxidase: in vitro and in vivo consequences of metal binding and oligomerization." available at J. Willard Marriott Library Special Collection. QP6.5 2007 .C69.
Rights Management © Harold Jerome Coyne III.
Format Medium application/pdf
Format Extent 5,640,342 bytes
Identifier undthes,4897
Source Original: University of Utah Spencer S. Eccles Health Sciences Library (no longer available).
Master File Extent 5,640,371 bytes
ARK ark:/87278/s60003z9
Setname ir_etd
Date Created 2012-04-24
Date Modified 2012-04-24
ID 191443
Reference URL https://collections.lib.utah.edu/ark:/87278/s60003z9