Conformational properties of cardiolipin-bound cytochrome c

Abstract

Interactions of cytochrome c (cyt c) with cardiolipin (CL) are important for both electron transfer and apoptotic functions of this protein. A sluggish peroxidase in its native state, when bound to CL, cyt c catalyzes CL peroxidation, which contributes to the protein apoptotic release. The heterogeneous CL-bound cyt c ensemble is difficult to characterize with traditional structural methods and ensemble-averaged probes. We have employed time-resolved FRET measurements to evaluate structural properties of the CL-bound protein in four dansyl (Dns)-labeled variants of horse heart cyt c. The Dns decay curves and extracted Dns-to-heme distance distributions PðrÞ reveal a conformational diversity of the CL-bound cyt c ensemble with distinct populations of the polypeptide structures that vary in their degree of protein unfolding. A fraction of the ensemble is substantially unfolded, with Dns-to-heme distances resembling those in the guanidine hydrochloride-denatured state. These largely open cyt c structures likely dominate the peroxidase activity of the CL-bound cyt c ensemble. Site variations in PðrÞ distributions uncover structural features of the CL-bound cyt c, rationalize previous findings, and implicate the prime role of electrostatic interactions, particularly with the protein C terminus, in the CL-induced unfolding.membrane | redox protein | fluorescence

The key role played by charge in the interaction of cytochrome c with cardiolipin

“…In the present study, CD Soret spectra show that the Lys72Arg and Lys73Arg mutants have structure similar to that of the wt protein, while the Lys72Asn and Lys73Asn mutants are the variants that most differ from it. The decreased rotational strength of the 414-nm band observed for the Asn mutants, is likely attributable to the presence of a minor heterogeneous subpopulation with non-native X-Fe-His18 axial coordination (where X is a misligated endogenous ligand) in equilibrium with the major Met80-Fe-His18 form [14,15,17]. Upon mutation, also the near-UV (270-300 nm) dichroic band decreases on passing from Arg to Ala to Asn (Fig.…”

“…The interaction with CL induces a heterogeneous ensemble of non-native protein species [14,15]. CL-bound cyt c shows altered tertiary structure, a perturbed heme crevice and the displacement of Met80 from the sixth coordination position of the heme-Fe atom [5][6][7][8][16][17][18][19].…”

Defining the Apoptotic Trigger

“…Most studies of this kind have used the exterior surface of liposome membranes to follow the interaction of cytochrome c with cardiolipin. An impressive number have been conducted on the mechanism of this unfolding phase (4,29,45,47,48) and this gross structural perturbation leads to the activation of the peroxidase activity that subsequently initiates critical modifications of cardiolipin (26,28,29,49,50).…”

“…Normally, cytochrome c exists in the mitochondrial inter-membrane space where it acts as an electron shuttle between complex III and complex IV, which reside in the inner mitochondrial membrane. The inner mitochrondrial membrane contains appreciable levels of the anionic phospholiplid cardiolipin (CL), 4 which is thought to interact with the highly basic cytochrome c. Considerable evidence has accrued to suggest that the interaction of cardiolipin with cytochrome c activates a normally suppressed peroxidase activity in this heme protein (2). Cardiolipin is subsequently specifically oxidized and it is postulated that the oxidation products facilitate the leakage of cytochrome c to the cytosol.…”

Role of Lysines in Cytochrome c–Cardiolipin Interaction

“…13 The CL-bound cyt c shows an altered tertiary structure and a perturbed heme crevice; furthermore, the native Met80−Fe(III) axial bond is lost. Depending on the conditions, the sixth coordination position of the heme iron remains unbound or is occupied by another side chain (possibly a lysine).…”