Purification and Characterization of the Cytochrome BC(1) Complex From Rhodobacter Sphaeroides

A highly active, large-scale preparation of ubiquinol:cytochrome $c\sb2$ oxidoreductase (cytochrome $bc\sb1$ complex) has been obtained from Rhodobacter sphaeroides. The enzyme was extracted from chromatophores using dodecyl maltoside in the presence of glycerol, and was purified by anion exchange and gel filtration chromatography. SDS-polyacrylamide gel electrophoresis showed the presence of four polypeptide subunits with apparent molecular weight values of 40, 34, 27, and 14 kDa. Values for ubiquinol, phospholipid, and iron content were determined, and the activity of the complex was shown to be enhanced in the presence of phospholipid. Full absorption spectrum redox titrations showed that the two thermodynamically distinct $b$-hemes and cytochrome $c\sb1$ possessed spectral features and redox midpoint potentials similar to those found in chromatophores. Redox titrations of the Rieske iron-sulfur center were monitored by low-temperature EPR spectroscopy, and its midpoint potential was shifted from +280 to +315 mV upon addition of inhibitor. The redox properties of the antimycin-sensitive ubiquinone species were also studied by EPR spectroscopy, and a pH dependence of $-$53 mV/pH unit was observed for the midpoint of the bound Q$\sb{\rm i}$/Q$\sb{\rm i}$H$\sb2$ couple. The results were very similar to those obtained in chromatophores. The purification procedure yielded at least 35 mg of pure cytochrome $bc\sb1$ complex from four grams of membrane protein, with a specific heme content of 8.9 nmol $c\sb1$ per mg protein, and consistently resulted in an enzyme preparation which catalyzed the reduction of horse heart cytochrome $c$ with a turnover in excess of 300 sec$\sp{-1}$.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2014

Author dc:creator

• Andrews, Katherine McKee

Contributors dc:contributor

• Gennis, Robert B.

• Chemistry, Biochemistry
• Biophysics, General

Identifier

(UMI)AAI8908609

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/70573