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A conserved steroid binding site in cytochrome C oxidase
Ling Qin1, Denise A Mills, Leann Buhrow
1Biochemistry and Molecular Biology Department, Michigan State University, East Lansing, Michigan 48824, USA.
Biochemistry
|September 2, 2008
Summary
Bile salt deoxycholate reactivates an inactive mutant of Rhodobacter sphaeroides cytochrome c oxidase. Structural analysis reveals deoxycholate binding at the K proton pathway entrance, suggesting a conserved regulatory site for steroids.
Area of Science:
- Biochemistry
- Structural Biology
- Enzyme Kinetics
Background:
- Rhodobacter sphaeroides cytochrome c oxidase is crucial for cellular respiration.
- The K proton pathway is essential for enzyme function.
- Mutations can inactivate enzyme activity, necessitating functional rescue studies.
Purpose of the Study:
- To investigate the effect of bile salts on an inactive mutant of Rhodobacter sphaeroides cytochrome c oxidase.
- To determine the binding site and mechanism of action of deoxycholate.
- To explore the potential for steroid-mediated regulation of the enzyme's K proton pathway.
Main Methods:
- Site-directed mutagenesis to create the E101A mutant.
- Enzyme activity assays to measure oxidase function.
- X-ray crystallography to determine the enzyme's structure with bound deoxycholate.
Main Results:
- Micromolar deoxycholate restored activity to the E101A mutant.
- Crystal structure showed deoxycholate bound at the K proton pathway entrance.
- The binding site is conserved and structurally similar to that of cholate in bovine oxidase.
Conclusions:
- Deoxycholate can functionally rescue an inactive cytochrome c oxidase mutant.
- A conserved steroid binding site at the K proton pathway entrance suggests a regulatory role.
- Steroids or related molecules may regulate proton translocation in cytochrome c oxidases.
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