RESUMO
The interaction between cardiolipin (CL) and cytochrome c (cyt-c) results in a gain of function of peroxidase activity by cyt-c. Despite intensive research, disagreements on nature and molecular details of this interaction remain. In particular, it is still not known how the interaction triggers the onset of apoptosis. Enzymatic characterization of peroxidase activity has highlighted the need for a critical threshold concentration of CL, a finding of profound physiological relevance in vivo. Using solution NMR, fluorescence spectroscopy, and in silico modeling approaches we here confirm that full binding of cyt-c to the membrane requires a CL:cyt-c threshold ratio of 5:1. Among three binding sites, the simultaneous binding of two sites, at two opposing sides of the heme, provides a mechanism to open the heme crevice to substrates. This results in "productive binding" in which cyt-c then sequesters CL, inducing curvature in the membrane. Membrane perturbation along with lipid peroxidation, due to interactions of heme/CL acyl chains, initiates the next step in the apoptotic pathway of making the membrane leaky. The third CL binding site while allowing interaction with the membrane, does not cluster CL or induce subsequent events, making this interaction "unproductive".
Assuntos
Cardiolipinas/metabolismo , Citocromos c/metabolismo , Membranas/metabolismo , Peroxidase/metabolismo , Sequência de Aminoácidos , Animais , Cardiolipinas/química , Citocromos c/química , Citocromos c/genética , Cavalos , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Peroxidase/química , Peroxidase/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Relação Estrutura-Atividade , Lipossomas UnilamelaresRESUMO
We attempted to evaluate the affinity of the anionic phospholipids to cytochrome c by means of surface plasmon resonance (SPR) technique and to correlate it with the cytochrome c active site alterations and peroxidase activity. Our experiments showed a strong interdependence between the phospholipid fatty acid saturation degree, the active site structure alterations and peroxidase activity of the cytochrome c phospholipid complex. Cytochrome c peroxidase activity and Trp59 fluorescence increase in the sequence of phosphatidyl choline (PC)-->phosphatidylserine (PS)-->cardiolipin (CL)-->phosphatidic acid (PA). The association constant (K(a)) increased in the sequence PC-->PA-->PS-->CL. The SPR spectroscopy data shows that K(a) is independent of lipid saturation degree, but correlates with phospholipid negative charge value.