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 P(r) 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 P(r) 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.

Fig. 1.

(A) Structure of horse heart cyt c showing the labeled positions. (B) Structure of the Dns label.

Fig. 2.

(A) Fluorescence anisotropy of ZnP (λ_ex = 423 nm, λ_em = 588 nm, [Zncyt] = 2 μM, and [cyt c] = 3 μM) with added TOCL/DOPC liposomes as a function of total lipid concentration. Results of multiple independent experiments have indicated that at 660 μM lipid, addition of 150 mM NaCl consistently leads to a decrease in the ZnP anisotropy value. The line is a fit to the Hill binding equation with K_a = 1.5 × 10⁴ M^-1. (B) CD spectra of cyt c ([cyt c] = 5 μM, l = 0.2 cm) in a 25 mM TES buffer at pH 7.4 with (CL-bound, blue) and without (native, black) TOCL/DOPC liposomes at 660 μM total lipid. (C) Fluorescence spectra (λ_ex = 295 nm) of cyt c and LysTrpLys in a 25 mM Hepes buffer at pH 7.4 with (cyt c—blue; LysTrpLys—cyan) and without (cyt c—black; LysTrpLys—magenta) TOCL/DOPC liposomes at 660 μM total lipid.

Fig. 3.

Fluorescence spectra (λ_ex = 336 nm) and distributions of D-A distances P(r) for four Dns-labeled variants of cyt c in a 25 mM Hepes buffer at pH 7.4 [native (N), gray]; with TOCL/DOPC liposomes at 660 μM total lipid [CL-bound (CL), blue]; with TOCL/DOPC liposomes at 660 μM total lipid and 150 mM NaCl (CL + salt, red); and in 5.8 ± 0.2 M GuHCl solution at pH 7.4 [GuHCl-unfolded (U_GuHCl), green]. The corresponding P(k) distributions are shown in Fig. S3. At distances longer than 1.5 × R₀ = 59 Å, energy transfer rate constants and D-A distances cannot be determined reliably; the structures with r≥1.5R₀ are represented by a single bar.

Fig. 4.

(A) Distributions of D-A distances P(r) from TR-FRET for Dns92 in the native state and with TOCL/DOPC liposomes at 660 μM total lipid at indicated concentrations of NaCl. (B) Peroxidase activity of cyt c in the native state and with TOCL/DOPC liposomes at 660 μM total lipid at indicated concentrations of NaCl, monitored with a guaiacol oxidation assay. (C) Initial rates versus population of extended cyt c structures (from Dns92 TR-FRET). A linear fit predicts an initial rate dA₄₇₀/dTime of 0.045 s^-1 at 100% of extended strucures, almost 100-fold slower than the value of 3.9 s^-1 determined experimentally for the protein in 6 M GuHCl at pH 7.4.

Fig. 5.

A cartoon illustrating populations of different cyt c conformations in the CL-bound ensemble.