Levi J. McClelland

Structure of a mitochondrial cytochrome c conformer competent for peroxidase activity

Significance Cytochrome c is essential to two important biochemical pathways, the electron transport chain and the intrinsic pathway of apoptosis. The heme crevice loop, which provides the Met80 ligand to the heme cofactor, is the most highly conserved segment of the cytochrome c sequence. The dynamics of this loop are likely important for both functions. Cytochrome c-mediated peroxidation of cardiolipin in the inner mitochondrial membrane is an early signal in apoptosis. We show that mutation of trimethyllysine 72 to alanine in yeast iso-1-cytochrome c allows formation of a conformer of the protein with Met80 displaced from the heme and enhances peroxidase activity. Thus, this residue is likely an important modulator of the peroxidase function of cytochrome c. At the onset of apoptosis, the peroxidation of cardiolipin at the inner mitochondrial membrane by cytochrome c requires an open coordination site on the heme. We report a 1.45-Å resolution structure of yeast iso-1-cytochrome c with the Met80 heme ligand swung out of the heme crevice and replaced by a water molecule. This conformational change requires modest adjustments to the main chain of the heme crevice loop and is facilitated by a trimethyllysine 72-to-alanine mutation. This mutation also enhances the peroxidase activity of iso-1-cytochrome c. The structure shows a buried water channel capable of facilitating peroxide access to the active site and of moving protons produced during peroxidase activity to the protein surface. Alternate positions of the side chain of Arg38 appear to mediate opening and closing of the buried water channel. In addition, two buried water molecules can adopt alternate positions that change the network of hydrogen bonds in the buried water channel. Taken together, these observations suggest that low and high proton conductivity states may mediate peroxidase function. Comparison of yeast and mammalian cytochrome c sequences, in the context of the steric factors that permit opening of the heme crevice, suggests that higher organisms have evolved to inhibit peroxidase activity, providing a more stringent barrier to the onset of apoptosis.

Disruption of a hydrogen bond network in human versus spider monkey cytochrome c affects heme crevice stability.

The hypothesis that the recent rapid evolution of primate cytochromes c, which primarily involves residues in the least stable Ω-loop (Ω-loop C, residues 40-57), stabilizes the heme crevice of cytochrome c relative to other mammals, is tested. To accomplish this goal, we have compared the properties of human and spider monkey cytochrome c and a set of four variants produced in the process of converting human cytochrome c into spider monkey cytochrome c. The global stability of all variants has been measured by guanidine hydrochloride denaturation. The stability of the heme crevice has been assessed with the alkaline conformational transition. Structural insight into the effects of the five amino acid substitutions needed to convert human cytochrome c into spider monkey cytochrome c is provided by a 1.15Å resolution structure of spider monkey cytochrome c. The global stability for all variants is near 9.0kcal/mol at 25°C and pH7, which is higher than that observed for other mammalian cytochromes c. The heme crevice stability is more sensitive to the substitutions required to produce spider monkey cytochrome c with decreases of up to 0.5 units in the apparent pKa of the alkaline conformational transition relative to human cytochrome c. The structure of spider monkey cytochrome c indicates that the Y46F substitution destabilizes the heme crevice by disrupting an extensive hydrogen bond network that connects three surface loops including Ω-loop D (residues 70-85), which contains the Met80 heme ligand.

Cytochrome c Can Form a Well-Defined Binding Pocket for Hydrocarbons.

Cytochrome c can acquire peroxidase activity when it binds to cardiolipin in mitochondrial membranes. The resulting oxygenation of cardiolipin by cytochrome c provides an early signal for the onset of apoptosis. The structure of this enzyme-substrate complex is a matter of considerable debate. We present three structures at 1.7-2.0 Å resolution of a domain-swapped dimer of yeast iso-1-cytochrome c with the detergents, CYMAL-5, CYMAL-6, and ω-undecylenyl-β-d-maltopyranoside, bound in a channel that places the hydrocarbon moieties of these detergents next to the heme. The heme is poised for peroxidase activity with water bound in place of Met80, which serves as the axial heme ligand when cytochrome c functions as an electron carrier. The hydroxyl group of Tyr67 sits 3.6-4.0 Å from the nearest carbon of the detergents, positioned to act as a relay in radical abstraction during peroxidase activity. Docking studies with linoleic acid, the most common fatty acid component of cardiolipin, show that C11 of linoleic acid can sit adjacent to Tyr67 and the heme, consistent with the oxygenation pattern observed in lipidomics studies. The well-defined hydrocarbon binding pocket provides atomic resolution evidence for the extended lipid anchorage model for cytochrome c/cardiolipin binding. Dimer dissociation/association kinetics for yeast versus equine cytochrome c indicate that formation of mammalian cytochrome c dimers in vivo would require catalysis. However, the dimer structure shows that only a modest deformation of monomeric cytochrome c would suffice to form the hydrocarbon binding site occupied by these detergents.

The response of Ω-loop D dynamics to truncation of trimethyllysine 72 of yeast iso-1-cytochrome c depends on the nature of loop deformation

Trimethyllysine 72 (tmK72) has been suggested to play a role in sterically constraining the heme crevice dynamics of yeast iso-1-cytochrome c mediated by the Ω-loop D cooperative substructure (residues 70–85). A tmK72A mutation causes a gain in peroxidase activity, a function of cytochrome c that is important early in apoptosis. More than one higher energy state is accessible for the Ω-loop D substructure via tier 0 dynamics. Two of these are alkaline conformers mediated by Lys73 and Lys79. In the current work, the effect of the tmK72A mutation on the thermodynamic and kinetic properties of wild-type iso-1-cytochrome c (yWT versus WT*) and on variants carrying a K73H mutation (yWT/K73H versus WT*/K73H) is studied. Whereas the tmK72A mutation confers increased peroxidase activity in wild-type yeast iso-1-cytochrome c and increased dynamics for formation of a previously studied His79-heme alkaline conformer, the tmK72A mutation speeds return of the His73-heme alkaline conformer to the native state through destabilization of the His73-heme alkaline conformer relative to the native conformer. These opposing behaviors demonstrate that the response of the dynamics of a protein substructure to mutation depends on the nature of the perturbation to the substructure. For a protein substructure which mediates more than one function of a protein through multiple non-native structures, a mutation could change the partitioning between these functions. The current results suggest that the tier 0 dynamics of Ω-loop D that mediates peroxidase activity has similarities to the tier 0 dynamics required to form the His79-heme alkaline conformer.

Structures of Ric-8A, a G protein chaperone and activator

Ric-8A is a 60 kDa protein expressed in the cytoplasm of multicellular eukaryotic cells. Unrelated to G protein-coupled receptors, Ric-8A has in vitro activity as a guanine nucleotide exchange factor. As such, Ric-8A catalyzes the release of GDP of GDP from the alpha subunits of heterotrimeric G proteins (Ga), forming a nucleotide-free Ga:Ric8A complex. In the presence of GTP or non-hydrolyzable GP analogs, the complex dissociates, releasing Ga•GTP and Ric-8A. Ric-8A controls the cellular abundance of Ga by inhibiting its ubiquitination, and acting as a chaperone. The mechanisms by which Ric-8A carries out these essential activities are unknown.