Interaction of Carbon Monoxide with the Apoptosis-Inducing Cytochrome c−Cardiolipin Complex

Abstract

The interaction of mitochondrial cytochrome (cyt) c with cardiolipin (CL) is involved in the initial stages of apoptosis. This interaction can lead to destabilization of the heme-Met80 bond and peroxidase activity [Basova, L. V., et al. (2007) Biochemistry 46, 3423-3434]. We show that under these conditions carbon monoxide (CO) binds to cyt c, with very high affinity ( approximately 5 x 10(7) M(-1)), in contrast to the native cyt c protein involved in respiratory electron shuttling that does not bind CO. Binding of CO to the cyt c-CL complex inhibits its peroxidase activity. Photodissociated CO from the cyt c-CL complex shows <20% picosecond geminate rebinding and predominantly bimolecular rebinding, with a second-order rate constant of approximately 10(7) M(-1) s(-1), an order of magnitude higher than in myoglobin. These findings contrast with those of Met80X mutant cyt c, where picosecond geminate recombination dominates due to the rigidity of the protein. Our data imply that CL leads to substantial changes in protein conformation and flexibility, allowing access of ligands to the heme. Together with the findings that (a) approximately 30 CL per cyt c are required for full CO binding and (b) salt-induced dissociation indicates that the two negative headgroup charges interact with approximately 5 positive surface charges of the protein, these results are consistent with a CL anchorage model with an acyl chain impaled in the protein [Kalanxhi, E., and Wallace, C. J. A. (2007) Biochem. J. 407, 179-187]. The affinity of CO for the complex is high enough to envisage an antiapoptotic effect of nanomolar CO concentrations via inhibition of the cyt c peroxidase activity.

Cardiolipin drives cytochrome c proapoptotic and antiapoptotic actions

“…CL binding to hhcytc induces a drastically reduced midpoint potential, which falls out of the range required for its physiological role in mitochondrial respiration (17). Further, the cleavage of the distal Fe-Met80 bond endows hhcytc with proapoptotic activity, due to the achievement of peroxidase action (15, 23-26, 31, 32), and antiapoptotic functions, increasing the affinity for CO and NO (27,28) and inducing peroxynitrite detoxification properties (29).…”

“…Antiapoptotic Activity of CL-bound Horse Heart Cytochrome c CL binding to hhcytc facilitates CO and NO binding to the heme-Fe-atom and peroxynitrite isomerization (27)(28)(29). While native ferrous hhcytc (hhcytc-Fe(II)) is unable to bind CO (35), ferrous CL-hhcytc (CL-hhcytc-Fe(II)), like mammalian myoglobins (Mbs) (36), interacts with CO by a simple second-order process (27).…”

“…While native ferrous hhcytc (hhcytc-Fe(II)) is unable to bind CO (35), ferrous CL-hhcytc (CL-hhcytc-Fe(II)), like mammalian myoglobins (Mbs) (36), interacts with CO by a simple second-order process (27). The value of the apparent dissociation equilibrium constant for CL-hhcytc-Fe(II) carbonylation ($3 3 10 28 M) (27) corresponds to that reported for ferrous horse heart Mb (Mb-Fe(II)) generally taken as a molecular model ($3 3 10 28 M) (36). However, both combination and dissociation rate shown.…”

“…The interaction with CL is pivotal for switching cytc function(s) from mitochondrial respiration to apoptosis. In fact, upon CL binding, cytc has been shown (i) to change its tertiary structure disrupting the hemeFe-Met80 distal bond (the proximal axial ligand being His) and, in some cases, to vary the spin state of the metal (4,14,(18)(19)(20)(21)(22), (ii) to reduce drastically the midpoint potential out of the range required for its role in the respiratory chain (17), (iii) to display peroxidase activity, using CL as a favorable substrate (15,(23)(24)(25)(26), (iv) to bind CO and NO with high affinity (27,28), and (v) to facilitate peroxynitrite scavenging (i.e., conversion to NO 3 2 ) (29). All these effects suggest that CL-bound cytc (CL-cytc) could play either proapoptotic effects, catalyzing lipid peroxidation, and cytc release into the cytoplasm, or antiapoptotic actions, scavenging peroxynitrite (i.e., protecting the mitochondrion from reactive nitrogen and oxygen species), and binding CO and NO (i.e., inhibiting lipid peroxidation and cytc traslocation).…”

“…2) (20). As a whole, CL binding to hhcytc seems to induce a gross conformational change(s) on both the proximal and distal side of the heme, causing the loss of the electron transfer properties of hhcytc and its transformation into a globin-and peroxidaselike heme-protein (15,17,(27)(28)(29).…”