Insights into cytochrome c-cardiolipin interaction. Role played by ionic strength.
The finding that cytochrome c (cyt c) plays a role in programmed cell death after its release from the mitochondrion has recently renewed interest in this protein. The structural changes in cytochrome c observed at early stages of the apoptotic process have been related to changes occurring in the protein when it forms a complex with phospholipid vesicles. Among the lipids constituting the membrane, cardiolipin is the one thought to bind to cyt c. In this paper, we have investigated the influence exerted by ionic strength on cytochrome c-cardiolipin interaction and found that formation of the cytochrome c-cardiolipin complex occurs via two distinct transitions, implying a high-affinity site and a low-affinity site. Ionic strength significantly influences complex stability; sodium chloride dissociates the complex through two distinct transitions, the second of which occurs at a very high anion concentration. ATP also dissociates the complex, but under the conditions that were investigated, its action is limited to the high-affinity site. The dissociation process is characterized by a very slow kinetic rate constant ( k obs = 4.2 x 10 (-3) s (-1)) and requires several minutes to be completed. We ascribe it to the high activation barrier met by the protein when restoring the native Fe(III)-M80 axial bond. The peroxidase activity shown by cardiolipin-bound cytochrome c is indicative of a less packed protein tertiary conformation in the complex. In line with earlier reports, these data highlight the manifold functions of cytochrome c besides the well-known role it plays in oxidative phosphorylation, shedding more light on the properties of the cytochrome c-cardiolipin complex, involved in the progression of early stages of apoptosis.
Sinibaldi
Federica
F
Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Università di Roma, Tor Vergata, 00133 Roma, Italy.
Fiorucci
Laura
L
Patriarca
Antonella
A
Lauceri
Rosaria
R
Ferri
Tommaso
T
Coletta
Massimo
M
Santucci
Roberto
R
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002308:Cardiolipins / Q000737:chemistry* / Q000378:metabolism*; D002417:Cattle; D002942:Circular Dichroism; D045304:Cytochromes c / Q000737:chemistry* / Q000378:metabolism*; D006736:Horses; D007700:Kinetics; D009994:Osmolar Concentration; D009195:Peroxidase / Q000378:metabolism; D011485:Protein Binding; D017186:Titrimetry
D002308:Cardiolipins; D000255:Adenosine Triphosphate; D045304:Cytochromes c; D009195:Peroxidase