Unravelling the Non-Native Low-Spin State of the Cytochrome c–Cardiolipin Complex: Evidence of the Formation of a His-Ligated Species Only

Abstract

The interaction between cytochrome c (Cyt c) and cardiolipin (CL) plays a vital role in the early stages of apoptosis. The binding of CL to Cyt c induces a considerable increase in its peroxidase activity that has been attributed to the partial unfolding of the protein, dissociation of the Met80 axial ligand, and formation of non-native conformers. Although the interaction between Cyt c and CL has been extensively studied, there is still no consensus regarding the conformational rearrangements of Cyt c that follow the protein-lipid interaction. To rationalize the different results and gain better insight into the Cyt c-CL interaction, we have studied the formation of the CL complex of the horse heart wild-type protein and selected mutants in which residues considered to play a key role in the interaction with CL (His26, His33, Lys72, Lys73, and Lys79) have been mutated. The analysis was conducted at both room temperature and low temperatures via ultraviolet-visible absorption, resonance Raman, and electron paramagnetic resonance spectroscopies. The trigger and the sequence of CL-induced structural variations are discussed in terms of disruption of the His26-Pro44 hydrogen bond. We unequivocally identify the sixth ligand in the partially unfolded, non-native low-spin state that Cyt c can adopt following the protein-lipid interaction, as a His ligation, ruling out the previously proposed involvement of a Lys residue or an OH ion.

Probing the non‐native states of Cytochrome c with resonance Raman spectroscopy: A tool for investigating the structure–function relationship

“…The His–Fe–OH − model has been obtained from the horse heart Cyt c Lys72Asn/Lys73Asn/Lys79Asn triple mutant at pH 12.0 . The RR spectra of this model are identical to those previously obtained for yeast iso‐1 Cyt c at pH > 10.5 and assigned to a deprotonated water bound to the heme iron .…”

“…The His18–Fe–Lys misligated form model compound has been obtained by preparing horse heart ferric Cyt c at pH 12, which gives rise to RR spectra almost identical to those previously reported for iso‐1 Cyt c at around pH 9.5 and is characterized by an Electron paramagnetic resonance (EPR) spectrum with typical Lys g 1 features near 3.5 and 3.3 . In the fingerprint region (Figure , right), the band at 384 cm −1 is assigned to the ν(Fe‐N Lys ) stretching vibration.…”

“…Absorption spectrum in the Soret region with its second derivative (top left) and resonance Raman (λ exc = 406.7 nm) spectra in the ν 3 (bottom left) and fingerprint (300–440 cm −1 ; bottom right) regions of native horse heart ferric Cyt c (wild type, axial coordination His–Fe–Met). Experimental conditions as reported in Milazzo et al…”

“…The Cyt c imidazole complex has been prepared following a previously published procedure . Briefly, an imidazole solution in sodium dodecyl sulfate was added to ferric Cyt c in phosphate buffer at pH 7.0 to obtain a final sodium dodecyl sulfate concentration of 10 mM and an imidazole: Cyt c ratio of 2,500:1 …”

“…Recently, the sequence of structural events that is prompted by progressive additions of CL to Cyt c has been monitored by electronic absorption, RR (at room temperature and 80 K), and EPR spectroscopies using horse heart Cyt c (Figure ) and specific site‐directed mutants. The RR characterization of the different misligated forms allowed the long‐standing debate about the nature of the structural changes of Cyt c induced by CL in the pre‐apoptotic process to be resolved . The main misligated species has been demonstrated to have His18–Fe–His coordination.…”