Electrostatic destabilization of the cytochrome b6f complex in the thylakoid membrane.
Three of the membrane-spanning polypeptides of the chloroplast cytochrome (cyt) b6f complex were sequentially released from the thylakoid membrane, in the order cyt b6, suIV and Rieske iron-sulfur protein, as the pH was increased from 10 to 12, a protocol usually employed to remove peripheral proteins from membranes. The fourth polypeptide of the cyt b6f complex, cyt f, which spans the membrane once, was apparently not released. The pH values for half-release at low ionic strength were approximately 10.7, 11.1 and 11.3 respectively. The separation of the polypeptides of the complex and the sequential release is readily seen at pH 11, where the loss from the membrane of cyt b6, suIV and Fe iron-sulfur center is approximately 90%, 50% and 20%, respectively. the release of cyt b6 from the membrane was reflected by the absence of its characteristic reduced minus oxidized absorbance signal. The pH values at which the release occurred increased as the ionic strength was raised, implying that the release of the b6f polypeptides arises from extrusion due to repulsive electrostatic interactions probably caused by deprotonation of tyrosine and lysine residues. The lipid content of the released polypeptides was very low, consistent with the observation of a non-membranous state. It is proposed that the pH-dependent extrusion requires two electrostatic effects at alkaline pH higher than approximately 10.5: (i) increased electrostatic repulsion between neighbouring polypeptides of the complex, arising from increased net negative charge in the peripheral segments of these polypeptides, which can cause separation of the polypeptides from the complex; and (ii) ionization of residues such as tyrosine in the membrane-spanning alpha-helices, and neutralization of residues such as lysine which can bind to the negative membrane surface.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907.
A
A
Szczepaniak
D
D
Huang
T W
TW
Keenan
W A
WA
Cramer
D015153:Blotting, Western; D002736:Chloroplasts / Q000378:metabolism; D003580:Cytochromes / Q000378:metabolism / Q000502:physiology*; D045348:Cytochromes f; D004591:Electrophoresis, Polyacrylamide Gel; D004594:Electrophysiology; D005227:Fatty Acids / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000201:enzymology*; D008055:Lipids / Q000032:analysis; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D045322:Photosynthetic Reaction Center Complex Proteins; D010944:Plants; D011487:Protein Conformation; D013057:Spectrum Analysis
D003580:Cytochromes; D005227:Fatty Acids; D008055:Lipids; D008565:Membrane Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; D045348:Cytochromes f