Cytochrome c impaled: investigation of the extended lipid anchorage of a soluble protein to mitochondrial membrane models

Abstract

Cyt c (cytochrome c) has been traditionally envisioned as rapidly diffusing in two dimensions at the surface of the mitochondrial inner membrane when not engaged in redox reactions with physiological partners. However, the discovery of the extended lipid anchorage (insertion of an acyl chain of a bilayer phospholipid into the protein interior) suggests that this may not be exclusively the case. The physical and structural factors underlying the conformational changes that occur upon interaction of ferrous cyt c with phospholipid membrane models have been investigated by monitoring the extent of the spin state change that result from this interaction. Once transiently linked by electrostatic forces between basic side chains and phosphate groups, the acyl chain entry may occur between two parallel hydrophobic polypeptide stretches that are surrounded by positively charged residues. Alteration of these charges, as in the case of non-trimethylated (TML72K) yeast cyt c and Arg91Nle horse cyt c (where Nle is norleucine), led to a decline in the binding affinity for the phospholipid liposomes. The electrostatic association was sensitive to ionic strength, polyanions and pH, whereas the hydrophobic interactions were enhanced by conformational changes that contributed to the loosening of the tertiary structure of cyt c. In addition to proposing a mechanistic model for the extended lipid anchorage of cyt c, we consider what, if any, might be the physiological relevance of the phenomenon.

The effects of ATP and sodium chloride on the cytochrome c–cardiolipin interaction: The contrasting behavior of the horse heart and yeast proteins

“…In equine cyt c the binding regions for ATP (i.e., the cluster formed by residues E62, K88, R91; see Fig. 8, panel A) and for CL (the M80-containing loop [4,6,23]) are very close. The observation that ATP hinders CL binding to the protein may find a rationale in the fact that while cyt c-bound ATP stabilizes the 'native' form of the protein, the cyt c-bound CL tends to destabilize it.…”

“…In healthy cells a portion of cyt c (15-20%) remains tightly bound to cardiolipin (CL), one of the phospholipids constituting the mitochondrial membrane [1,2]. CL-bound cyt c shows tertiary structural rearrangements, which include alteration of the heme pocket region and detachment of M80 from the sixth coordination position of the heme iron [3][4][5][6]. The CL-specific peroxidase action acquired by membrane-bound cyt c in the early stages of apoptosis, which initiates the protein pro-apoptotic activity, is critical for cells; CL peroxidation induces cyt c release into the cytosol and favors the accumulation of products releasing pro-apoptotic factors [7][8][9][10].…”

“…Both consider that, upon binding, one acyl chain of CL protrudes in the protein interior. However, one model identifies the hosting region in the hydrophobic channel close to the invariant residue N52 [3], whereas the other in the M80-containing loop [4]. The fact that the CL liposomes bind cyt c at two distinct binding sites of the protein [5,11] led to the proposal that two acyl chains of CL (instead of one, as suggested by the two above mentioned models) may protrude into cyt c [11].…”

Role of Lysines in Cytochrome c–Cardiolipin Interaction

“…7 To this end, the Lys72Asn, Lys73Asn, Lys79Asn, Lys72/73Asn, and Lys72/73/79Asn mutants of horse heart cyt c were produced and characterized. Through the Lys → Asn mutation, positively charged residues are replaced with polar side chains carrying no net charge, under the conditions investigated.…”

“…It is proposed that the cleft formed by these side chains permits penetration of the acyl chain deep into the protein until it reaches the heme pocket region. 7 This model represents a plausible alternative to the extended lipid conformation model described above. 19 Very recently, the hypothesis that two acyl chains of CL, instead of one, may penetrate inside the protein during complex formation has been formulated.…”