Electron gating and mechanism of proton exit in quinol oxidation in cyt bc1 complex from R. sphaeroides

Victoria, Doreen C.

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https://hdl.handle.net/2142/16129 Copy

Description

Title

Electron gating and mechanism of proton exit in quinol oxidation in cyt bc1 complex from R. sphaeroides

Author(s)

Victoria, Doreen C.

Issue Date

2010-05-19T18:37:13Z

Director of Research (if dissertation) or Advisor (if thesis)

Crofts, Antony R.

Doctoral Committee Chair(s)

Crofts, Antony R.

Committee Member(s)

• Gennis, Robert B.
• Nair, Satish K.
• Spies, Maria

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Cytochrome bc1 complex
• electron gating mechanism of cytochrome bc1 complex
• proton exit mechanism of quinol oxidation of cytochrome bc1 complex
• quinol oxidation at the Qo-site
• second electron transfer of quinol oxidation of the bc1 complex
• inter-monomer electron transfer of cyt bc1 complex
• bL hemes inter-monomer electron transfer in cyt bc1 complex
• pre-steady state kinetics of quinol oxidation at the Qo-site of cyt bc1 complex from R. sphaeoroides
• in situ pre-steady state kinetics of cyt bc1 complex in R. sphaeroides
• Modified Q-cycle mechanism of Cytochrome bc1 complex
• Glu-295 (Glu-271 in bovine or Glu-272) of PEWY loop mutational studies of Cyt bc1 complex
• bypass reaction rates in cyt bc1 complex

Abstract

The cytochrome bc1 complex of Rhodobacter sphaeroides in wild-type and several strains mutated at the conserved -PEWY- glutamate residue (E272 in chicken, E295 in R. sphaeoroides) were characterized in situ to determine the functional role of this residue in the second electron transfer of quinol oxidation at the Qo-site. Previous studies showed that mutation at this position resulted to a substantially inhibited electron transfer, while the bypass rates were equal to or less than the wild-type strain. In addition, these strains showed resistance to stigmatellin and a modest increase in the Km. In this research, the effects of mutation at E295 on the following parameters have been studied: catalytic constant, Km; apparent pK values determining rate; binding constants for the enzyme-substrate complex, [ES]; the bypass reactions mediated through the transient semiquinone intermediate at the Qo-site; pH dependence of Km; and the thermodynamic properties of redox cofactors. Our results could be interpreted in terms of two roles for the -PEWY- glutamate: (i) in facilitating electron transfer from semiquinone (QH∙) to heme bL (low potential b-type heme of cyt b to generate the product quinone (Q); and (ii) in catalyzing both the deprotonation of QH∙ to semiquinone anion, Q∙ ‾, and exit of the proton via a water channel to the exterior. It has been suggested that inter-monomer electron transfer between the cytochrome bL hemes of the bc1 complex dimer is an essential feature of normal turnover. This functional role of the dimeric structure is suggested by the relatively short distance between the heme bL centers. Although a structural role in aligning the mobile extrinsic domain of the Rieske iron sulfur protein (ISP) with catalytic interfaces on cyt b and cyt c1 is now well established on the basis of the X-ray crystallographic structures and mutational studies, most previous discussions of mechanism have been in the context of a monomeric scheme. We have tested for inter-monomer electron transfer by assaying quinol turnover through reduction of cytochrome bH heme in the presence of antimycin, and by titrating the Qo-site with myxothiazol inhibitor. If inter-monomer electron transfer could occur at the rate suggested by Moser-Dutton-Marcus analysis, strongly bowed titration curves would be expected, which could be readily measured experimentally. Our results show simple linear titration curves, which are not compatible with claims from literature regarding the observation of inter-monomer electron transfer between the bL hemes.

Graduation Semester

2010-5

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http://hdl.handle.net/2142/16129

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Copyright 2010 Doreen C. Victoria

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