Multiple conformations of physiological membrane-bound cytochrome c.
One-tenth of cytochrome c (cyt c) remains bound to the inner mitochondrial membrane (IMM) at physiological ionic strength (I; i.e. , I approximately 150 mM), exhibiting decreased electron transport (ET) activity. We now show that this form of membrane-bound cyt c (MB-cyt c) can be obtained in vitro and that binding to membranes at low I generates an additional conformation with higher ET activity. This low I bound form of MB-cyt c (MBL-cyt c) exhibited intrinsic ET rates similar to those of electrostatically bound cyt c (EB-cyt c). The ET activity of IMM-bound MB-cyt c approached slowly that of MBL-cyt c or EB-cyt c, suggesting that MB-cyt c converts to MBL-cyt c while bound to IMM. When maintained at physiological I, both forms of MB-cyt c were released from the membrane, indicating that they convert to an EB-cyt c-like form. This process may be very dynamic in cellular mitochondria, as binding and release for both MB-cyt c forms increased considerably with temperature. I-Dependent binding of MB-cyt c does not require IMM, and it can be reproduced using large or small unilamellar vesicles (SUV). Using SUV-cyt c complexes, we characterized the secondary structure of MB-cyt c and MBL-cyt c by circular dichroism. Conformational analysis revealed that cyt c binding as MB-cyt c decreases its alpha-helical content (70-79%) and increases its beta-sheet up to 135%. The secondary structure of MBL-cyt c was similar to that of EB-cyt c and soluble cyt c, with a modest increase in beta-sheet. Taken together, our experiments suggest that physiological cyt c exists in soluble and membrane-bound conformations with similar ET activity, which may exchange very rapidly, and that soluble hydrophilic proteins can bind transiently to biomembranes.
Cortese
J D
JD
Department of Cell Biology and Anatomy and Laboratories for Cell Biology, The School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7090, USA.
Voglino
A L
AL
Hackenbrock
C R
CR
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000502:physiology*; D004579:Electron Transport; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012756:Sheep; D012995:Solubility; D013696:Temperature
D003574:Cytochrome c Group