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A Reexamination of Tryptophan Fluorescence in Cytochrome c Oxidase

Citation

Smith, Paul Andrew (1991) A Reexamination of Tryptophan Fluorescence in Cytochrome c Oxidase. Master's thesis, California Institute of Technology. http://resolver.caltech.edu/CaltechTHESIS:04192011-145437221

Abstract

Cytochrome c oxidase links reduction of oxygen with the pumping of protons across the inner mitochondrial membrane. Recently it has been shown that only two of the four electron transfers to the oxygen binding site are coupled with proton translocation. The discovery implies that there is a conformational switch between pumping and non-conformational changes. Gating conformational transitions occur in each cycle of the pump when electrons and protons are alternatively accessible to either the inside or outside of the membrane. Such transitions are linked to the redox state of the coupling site. The unloaded/loaded transition, on the other hand, depends on the intermediate at the oxygen binding site and is not part of the pumping cycle. A 16 nm red-shift in the tryptophan emission maximum of cytochrome oxidase upon reduction of the enzyme with dithionite was reported earlier [Copeland, R.A., Smith, P.A., and Chan, S.I. (1987) Biochemistry 26, 7311]. As such redox-linked change could be an important probe of pumping transitions, the fluorescence change of cytochrome oxidase upon reduction with ascorbate and cytochrome c was measured. The absence of a shift led to the reevalution of the earlier experiments for inner filter effects. The earlier experiments were reproduced with proper optical controls, and a method for reconstructing absorbance aftifacts from the reported fluourescence spectra was devised. Tryptophan fluourescence lifetimes were also measured. All the data support the conclusion that the earlier reports of a redox-linked fluourescence change were artifacts and that the other transient changes in fluorescence which have been measured cannot be associated with either the unloaded/loaded transition or a gating transition.

Item Type:Thesis (Master's thesis)
Subject Keywords:Chemistry
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Restricted to Caltech community only
Research Advisor(s):
  • Khundkar, Lutfur R. (advisor)
  • Perry, Joseph W. (advisor)
Thesis Committee:
  • Unknown, Unknown
Defense Date:31 May 1991
Record Number:CaltechTHESIS:04192011-145437221
Persistent URL:http://resolver.caltech.edu/CaltechTHESIS:04192011-145437221
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:6342
Collection:CaltechTHESIS
Deposited By: Benjamin Perez
Deposited On:19 Apr 2011 22:31
Last Modified:26 Dec 2012 04:34

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