Phospholipase digestion of bound cardiolipin reversibly inactivates bovine cytochrome bc1

Baltazar Gomez, Neal C. Robinson

Research output: Contribution to journalArticlepeer-review

118 Scopus citations

Abstract

Phospholipids and tightly bound cardiolipin (CL) can be removed from Tween 20 solubilized bovine cytochrome bc1 (EC 1.10.2.2) by digestion with Crotalus atrox phospholipase A2. The resulting CL-free enzyme exhibits all the spectral properties of native cytochrome bc1, but is completely inactive. Full electron transfer activity is restored by exogenous cardiolipin added in the presence of dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE), but not by cardiolipin alone or by mixtures of phospholipids lacking cardiolipin. Acidic, nonmitochondrial phospholipids, e.g., monolysocardiolipin or phosphatidylglycerol, partially reactivate CL-free cytochrome bc1 if they are added together with DOPC and DOPE. Phospholipid removal from the Tween 20 solubilized enzyme, including the tightly bound cardiolipin, does not perturb the environment of either cytochrome b562 or b566, nor does it cause the autoreduction of cytochrome c1. Cardiolipin-free cytochrome bc1 also binds antimycin and myxothiazol normally with the expected red shifts in b562 and b566, respectively. However, the CL-free enzyme is much less stable than the lipid- rich preparation, i.e., (1) many chromatographic methods perturb both cytochrome b566 (manifested by a hypsochromic effect, i.e., blue shift of 1.5-1.7 nm) and cytochrome c1 (evidenced by autoreduction in the absence of reducing agents); (2) affinity chromatographic purification of the enzyme causes pronounced loss of subunits VII and XI (65-80% decrease) and less significant loss of subunits I, IV, V, and X (20-30% decrease); and (3) high detergent-to-protein ratios result in disassembly of the complex. We conclude that the major role of the phospholipids surrounding cytochrome bc1, especially cardiolipin, is to stabilize the quaternary structure. In addition, bound cardiolipin has an additional functional role in that it is essential for enzyme activity.

Original languageEnglish (US)
Pages (from-to)9031-9038
Number of pages8
JournalBiochemistry
Volume38
Issue number28
DOIs
StatePublished - Jul 13 1999

ASJC Scopus subject areas

  • Biochemistry

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