Analysis of heme-copper ligation, quinol activity, and ligand binding kinetics of cytochrome BO(3) quinol oxidase from E. coli
Rumbley, Jon Nolan
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Permalink
https://hdl.handle.net/2142/20492
Description
Title
Analysis of heme-copper ligation, quinol activity, and ligand binding kinetics of cytochrome BO(3) quinol oxidase from E. coli
Author(s)
Rumbley, Jon Nolan
Issue Date
1995
Doctoral Committee Chair(s)
Gennis, Robert B.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Chemistry, Physical
Language
eng
Abstract
Structural and functional properties of wild type and mutant forms of cytochrome $bo\sb3$ quinol oxidase from E.coli were examined. Structural properties of subunit II were addressed with the restoration of the putative Cu$\sb{\rm A}$ ligands and construction of a chimeric E.coli/R.sphaeroides subunit II. Functional properties of subunit II were interpreted with azido-Q labelling studies. General properties of the wild type enzyme were interpreted with respect to heme content, ubiquinol content, absolute absorption spectra, ligand bound absorption spectra, and ligand binding kinetics. Structural and functional perterbations by the mutation of conserved residues were determined with ligand binding kinetics. Finally, construction and characterization of histidine tagged subunits I, II, and III was described.
The Cu$\sb{\rm A}$ site in the hydrophilic domain of cytochrome c oxidases is composed of ligands His, Cys, Cys, His, and Met and a conserved Glu appears between the two Cys. These residues were introduced into cytochrome $bo\sb3$ oxidase. The mutant was shown to bind two copper atoms as normal Cu$\sb{\rm A}$. Presence of the Cu$\sb{\rm A}$ site did not confer cytochrome c activity or disrupt ubiquinol activity. The subunit II chimeric construct containing the hydrophilic domain of R.sphaeroides and the hydrophobic domain of E.coli was simply proteolyzed. The location of the ubiquinol substrate binding site was determined with photoreactive substrate azido-Q which labels subunit II preferentially.
"Overexpressing strains contain high levels of $oo\sb3$ enzyme which lead to the split alpha absorption band. A ""good"" $bo\sb3$ preparation showed only a broad absorption at 562nm and Soret maxima at 407.5nm corresponding to ""fast"" cyanide binding form. Spectra of ligand bound forms of ""good"" $bo\sb3$ and kinetics of ligand binding were measured. Carbon monoxide recombination rates were determined for mutants of the seven conserved histidine residues and saturation kinetics determined for helix VI and VIII mutants."
"Finally, the construction and characterization of histidine tagged subunits I, II, and III was described. The subunit I and II constructs have wild type activities while the subunit III construct was unable to compliment aerobic growth. The subunit II construct enhanced the ratio of $bo\sb3/oo\sb3$ enzyme and consistently produced ""good"" enzyme while the subunit I construct was more heterogeneous."
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