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Lys-ligated cytochromes make up an emerging family of heme proteins. Density functional theory calculations on the amine/imidazole-ligated c-type ferric heme were employed to develop force-field parameters for molecular dynamics (MD) simulations of structural and dynamic features of these proteins. The new force-field parameters were applied to the alkaline form of yeast iso-1 cytochrome c to rationalize discrepancies resulting from distinct experimental conditions in prior structural studies and to provide insights into the mechanisms of the alkaline transition. Our simulations have revealed the dynamic nature of Ω-loop C in the Lys-ligated protein and its unfolding in the Lys-ligated conformer having this loop in the same position as in the native Met-ligated protein. The proximity of Tyr67 or Tyr74 to the Lys ligand of ferric heme iron suggests a possible mechanism of the backward alkaline transition where a proton donor Tyr assists in Lys dissociation. The developed force-field parameters will be useful in structural and dynamic characterization of other native or engineered Lys-ligated heme proteins. Show less

A 2.08 Å structure of an alkaline conformer of the domain-swapped dimer of K72A human cytochrome c (Cytc) crystallized at pH 9.9 is presented. In the structure, Lys79 is ligated to the heme. All other domain-swapped dimer structures of Cytc have water bound to this coordination site. Part of Ω-loop D (residues 70-85) forms a flexible linker between the subunits in other Cytc domain-swapped dimer structures but instead converts to a helix in the alkaline conformer of the dimer combining with the C-terminal helix to form two 26-residue helices that bracket both sides of the dimer. The alkaline transition of the K72A human dimer monitored at both 625 nm (high spin heme) and 695 nm (Met80 ligation) yields midpoint pH values of 6.6 and 7.6, respectively, showing that the Met80 → Lys79 and high spin to low spin transitions are distinct. The dimer peroxidase activity increases rapidly below pH 7, suggesting that population of the high spin form of the heme is what promotes peroxidase activity. Comparison of the structures of the alkaline dimer and the neutral pH dimer shows that the neutral pH conformer has a better electrostatic surface for binding to a cardiolipin-containing membrane and provides better access for small molecules to the heme iron. Given that the pH of mitochondrial cristae ranges from 6.9 to 7.2, the alkaline transition of the Cytc dimer could provide a conformational switch to tune the peroxidase activity of Cytc that oxygenates cardiolipin in the early stages of apoptosis. Show less

The A51V variant of human cytochrome c is linked to thrombocytopenia 4 (THC4), a condition that causes decreased blood platelet counts. A 1.82 Å structure of the A51V variant shows only minor changes in tertiary structure relative to the wild-type (WT) protein. Guanidine hydrochloride denaturation demonstrates that the global stability of the A51V variant is 1.3 kcal/mol less than that of the WT protein. The midpoint pH, pH_1/2, of the alkaline transition of the A51V variant is 1 unit less than that of the WT protein. Stopped-flow pH jump experiments show that the A51V substitution affects the triggering ionization for one of two kinetically distinguishable alkaline conformers and enhances the accessibility of a high-spin heme transient. The pH_1/2 for acid unfolding of the A51V variant is 0.7 units higher than for that of the WT protein. Consistent with the greater accessibility of non-native conformers for the A51V variant, the k_cat values for its peroxidase activity increase by 6- to 15-fold in the pH range of 5-8 versus those of the WT protein. These data along with previously reported data for the other THC4-linked variants, G41S and Y48H, underscore the role of Ω-loop C (residues 40-57) in modulating the peroxidase activity of cytochrome c early in apoptosis. Show less

Naturally occurring mutations found in one of the two Ω-loop substructures in human cytochrome c are associated with low blood platelet count (thrombocytopenia). Both Ω-loops participate in the formation of conformers associated with cytochrome c peroxidase activity and apoptotic function. At alkaline pH values, the Met80 ligand to the ferric heme iron dissociates, and a lysine residue in the 71-85 Ω-loop coordinates to the iron. The alkaline isomerization has been the focus of extensive kinetic studies, and it is established that a deprotonation triggers the release of the Met80 ligand (p K_trigger). A second deprotonation stabilizes a pentacoordinate heme form (p K_a2). In this study, site-directed variants at the 41 and 48 positions in the 40-57 Ω-loop and at the 81 and 83 positions in the 71-85 Ω-loop reveal that conformational transitions in the 71-85 Ω-loop, leading to the alkaline or peroxidatic conformers, are controlled by the 40-57 Ω-loop. We find that the variants causing thrombocytopenia, G41S and Y48H, lower the p K_trigger and increase p K_a2. Our results are presented in a mechanistic framework, depicted by a cube, that accounts for the pH dependencies of the equilibrium and kinetic parameters governing the alkaline transition of the native protein and Ω-loop variants. The data are most consistent with the trigger for Met80 replacement by a lysine being a deprotonation within a hydrogen bonded unit that links the two Ω-loops rather than an individual group. Such a proposal aligns with the entatic contribution made by the same unit in controlling the Met80-Fe(III) bond strength. Show less

Cytochrome c binds to cardiolipin (CL) on the inner mitochondrial membrane during the initial stages of apoptosis where it oxidizes CL, promoting its release into the cytoplasm where it initiates apoptosis. Previous work has identified interaction sites on cytochrome c involved in the cytochrome c-CL interaction. The contributions of the lysines attributed to site A, the anionic site, are studied here to elucidate the relative importance of each for electrostatic interaction of cytochrome c with CL at pH 8, conditions where site A is dominant. A set of single, double, and quadruple lysine to alanine variants of yeast iso-1-cytochrome c, at sequence positions 72, 73, 86, and 87, show that all contribute to the site A-mediated interaction with CL. All variants experience two sequential structural rearrangements as the lipid to protein ratio (LPR) increases. At a low LPR near 10, all variants undergo a small heme-centered structural change detected by Soret circular dichroism. At higher LPRs ranging from 22 to 34, all variants partially unfold as detected by Trp59 emission. The robustness of the mechanism of interaction to sequential neutralization of the four lysines assigned to site A demonstrates that site A is more extensive than previously supposed. The nature of both structural rearrangements also depends on which lysines constitute site A. The peroxidase activity of cytochrome c in the early stages of apoptosis depends on the nature of structural rearrangement near the heme. Thus, the lysines that comprise site A may have evolved to optimize the peroxidase signaling switch. Show less

The two roles of cytochrome c (cyt c), in oxidative phosphorylation and apoptosis, critically depend on redox properties of its heme iron center. The K79G mutant has served as a parent protein for a series of mutants of yeast iso-1 cyt c. The mutation preserves the Met80 coordination to the heme iron, as found in WT* (K72A/C102S), and many spectroscopic properties of K79G and WT* are indistinguishable. The K79G mutation does not alter the global stability, fold, rate of Met80 dissociation, or thermodynamics of the alkaline transition (p K_a) of the protein. However, the reduction potential of the heme iron decreases; further, the p K_H of the trigger group and the rate of the Met-to-Lys ligand exchange associated with the alkaline transition decrease, suggesting changes in the environment of the heme. The rates of electron self-exchange and bimolecular electron transfer (ET) with positively charged inorganic complexes increase, as does the intrinsic peroxidase activity. Analysis of the reaction rates suggests that there is increased accessibility of the heme edge in K79G and supports the importance of the Lys79 site for bimolecular ET reactions of cyt c, including those with some of its native redox partners. Structural modeling rationalizes the observed effects to arise from changes in the volume of the heme pocket and solvent accessibility of the heme group. Kinetic and structural analyses of WT* characterize the properties of the heme crevice of this commonly employed reference variant. This study highlights the important role of Lys79 for defining functional redox properties of cyt c. Show less

Met80, one of the heme iron ligands in cytochrome c (cyt c), is readily oxidized to Met sulfoxide (Met-SO) by several biologically relevant oxidants. The modification has been suggested to affect both the electron-transfer (ET) and apoptotic functions of this metalloprotein. The coordination of the heme iron in Met-oxidized cyt c (Met-SO cyt c) is critical for both of these functions but has remained poorly defined. We present electronic absorption, NMR, and EPR spectroscopic investigations as well as kinetic studies and mutational analyses to identify the heme iron ligands in yeast iso-1 Met-SO cyt c. Similar to the alkaline form of native cyt c, Lys73 and Lys79 ligate to the ferric heme iron in the Met80-oxidized protein, but this coordination takes place at much lower pH. The ferrous heme iron is ligated by Met-SO, implying the redox-linked ligand switch in the modified protein. Binding studies with the model peptide microperoxidase-8 provide a rationale for alterations in ligation and for the role of the polypeptide packing in native and Met-SO cyt c. Imidazole binding experiments have revealed that Lys dissociation from the ferric heme in K73A/K79G/M80K (M80K^#) and Met-SO is more than 3 orders of magnitude slower than the opening of the heme pocket that limits Met80 replacement in native cyt c. The Lys-to-Met-SO ligand substitution gates ET of ferric Met-SO cyt c with Co(terpy)₂²⁺. Owing to the slow Lys dissociation step, ET reaction is slow but possible, which is not the case for nonswitchable M80A and M80K^#. Acidic conditions cause Lys replacement by a water ligand in Met-SO cyt c (p K_a = 6.3 ± 0.1), increasing the intrinsic peroxidase activity of the protein. This pH-driven ligand switch may be a mechanism to boost peroxidase function of cyt c specifically in apoptotic cells. Show less

Cytochrome c (cyt c) is a small soluble heme protein characterized by a relatively flexible structure, particularly in the ferric form, such that it is able to sample a broad conformational space. Depending on the specific conditions, interactions, and cellular localization, different conformations may be stabilized, which differ in structure, redox properties, binding affinities, and enzymatic activity. The primary function is electron shuttling in oxidative phosphorylation, and is exerted by the so-called native cyt c in the intermembrane mitochondrial space of healthy cells. Under pro-apoptotic conditions, however, cyt c gains cardiolipin peroxidase activity, translocates into the cytosol to engage in the intrinsic apoptotic pathway, and enters the nucleus where it impedes nucleosome assembly. Other reported functions include cytosolic redox sensing and involvement in the mitochondrial oxidative folding machinery. Moreover, post-translational modifications such as nitration, phosphorylation, and sulfoxidation of specific amino acids induce alternative conformations with differential properties, at least in vitro. Similar structural and functional alterations are elicited by biologically significant electric fields and by naturally occurring mutations of human cyt c that, along with mutations at the level of the maturation system, are associated with specific diseases. Here, we summarize current knowledge and recent advances in understanding the different structural, dynamic, and thermodynamic factors that regulate the primary electron transfer function, as well as alternative functions and conformations of cyt c. Finally, we present recent technological applications of this moonlighting protein. Show less

Cytochromes (cyts) are ubiquitous heme containing proteins that are key components of energy transduction pathways. They participate in a wide variety of electron transfer reactions, which are essential for cellular processes responsible for chemical energy (ATP)... Show less