Perturbations in protein structure define the mechanism of allosteric regulation and biological information transfer. In cytochrome c (cyt c), ligation of Met80 to the heme iron is critical for the protein’s electron-transfer (ET) function in oxidative phosphorylation and for suppressing its peroxidase activity in apoptosis. The hard base Lys is a better match for the hard ferric iron than the soft base Met is, suggesting the key role of the protein scaffold in favoring Met ligation. To probe the role of the protein structure in the maintenance of Met ligation, mutations T49V and Y67R/M80A were designed to disrupt hydrogen bonding and packing of the heme coordination loop, respectively. Electronic absorption, nuclear magnetic resonance, and electron paramagnetic resonance spectra reveal that ferric forms of both variants are Lys-ligated at neutral pH. A minor change in the tertiary contacts in T49V, away from the heme coordination loop, appears to be sufficient to execute a change in ligation, suggesting a cross-talk between the different regions of the protein structure and a possibility of built-in conformational switches in cyt c. Analyses of thermodynamic stability, kinetics of Lys binding and dissociation, and the pH-dependent changes in ligation provide a detailed characterization of the Lys coordination in these variants and relate these properties to the extent of structural perturbations. The findings emphasize the importance of the hydrogen-bonding network in controlling ligation of the native Met80 to the heme iron.

It has been suggested that the alkaline form of cytochrome c (cyt c) regulates function of this protein as an electron carrier in oxidative phosphorylation and as a peroxidase that reacts with cardiolipin (CL) during apoptosis. In this form, Met80, the native ligand to the heme iron, is replaced by a Lys. While it has become clear that the structure of cyt c changes, the extent and sequence of conformational rearrangements associated with this ligand replacement remain a subject of debate. Herein we report a high-resolution crystal structure of a Lys73-ligated cyt c conformation that reveals intricate change in the heme environment upon this switch in the heme iron ligation. The structure is surprisingly compact, and the heme coordination loop refolds into a β-hairpin with a turn formed by the highly conserved residues Pro76 and Gly77. Repositioning of residue 78 modifies the intraprotein hydrogen-bonding network and, together with adjustments of residues 52 and 74, increases the volume of the heme pocket to allow for insertion of one of the CL acyl moieties next to Asn52. Derivatization of Cys78 with maleimide creates a solution mimic of the Lys-ligated cyt c that has enhanced peroxidase activity, adding support for a role of the Lys-ligated cyt c in the apoptotic mechanism. Experiments with the heme peptide microperoxidase-8 and engineered model proteins provide a thermodynamic rationale for the switch to Lys ligation upon perturbations in the protein scaffold.

Measurements of photoinduced Fe2+-to-Ru3+ electron transfer (ET), supported by theoretical analysis, demonstrate that mutations off the dominant ET pathways can strongly influence the redox reactivity of cytochrome c. The effects arise from the change in the protein dynamics mediated by the intraprotein hydrogen-bonding network.

Cardiolipin (CL) is a unique phospholipid found in mitochondrial inner membrane. It is a key component for mitochondrial function in both respiration and apoptosis. The level of CL is an important parameter for investigating these intracellular events and is a critical indicator of a number of diseases associated with mitochondrial respiratory functions. 10-Nonyl acridine orange (NAO) is the only fluorescent dye currently available for CL detection. However, the performance of NAO is far from satisfactory in terms of selectivity and sensitivity. In this work, we report an aggregation-induced emission-active fluorogen, TTAPE-Me, for CL detection and quantification. With improved sensitivity and excellent selectivity to CL over other major mitochondrial membrane lipids, TTAPE-Me could serve as a valuable fluorescent sensor for CL quantification. The use of TTAPE-Me for the quantification of isolated mitochondria is also demonstrated.

•Cyt c binding to CL membranes promotes dissociation of Met80 and opening of the heme crevice.•Many powerful spectroscopic probes have been employed to study cyt c–CL interactions.•Three different modes of cyt c interactions with CL membranes have been proposed.•The heterogeneous CL-bound cyt c ensemble consists of species that vary in the degree of protein unfolding.•Experimental conditions have a strong influence on the species populating the CL-bound cyt c ensemble.Interactions of cytochrome c (cyt c) with cardiolipin (CL) play a critical role in early stages of apoptosis. Upon binding to CL, cyt c undergoes changes in secondary and tertiary structure that lead to a dramatic increase in its peroxidase activity. Insertion of the protein into membranes, insertion of CL acyl chains into the protein interior, and extensive unfolding of cyt c after adsorption to the membrane have been proposed as possible modes for interaction of cyt c with CL. Dissociation of Met80 is accompanied by opening of the heme crevice and binding of another heme ligand. Fluorescence studies have revealed conformational heterogeneity of the lipid-bound protein ensemble with distinct polypeptide conformations that vary in the degree of protein unfolding. We correlate these recent findings to other biophysical observations and rationalize the role of experimental conditions in defining conformational properties and peroxidase activity of the cyt c ensemble. Latest time-resolved studies propose the trigger and the sequence of cardiolipin-induced structural transitions of cyt c.

Cysteine-bound hemes are key components of many enzymes and biological sensors. Protonation (deprotonation) of the Cys ligand often accompanies redox transformations of these centers. To characterize these phenomena, we have engineered a series of Thr78Cys/Lys79Gly/Met80X mutants of yeast cytochrome c (cyt c) in which Cys78 becomes one of the axial ligands to the heme. At neutral pH, the protonation state of the coordinated Cys differs for the ferric and ferrous heme species, with Cys binding as a thiolate and a thiol, respectively. Analysis of redox-dependent stability and alkaline transitions of these model proteins, as well as comparisons to Cys binding studies with the minimalist heme peptide microperoxidase-8, demonstrate that the protein scaffold and solvent interactions play important roles in stabilizing a particular Cys–heme coordination. The increased stability of ferric thiolate compared with ferrous thiol arises mainly from entropic factors. This robust cyt c model system provides access to all four forms of Cys-bound heme, including the ferric thiol. Protein motions control the rates of heme redox reactions, and these effects are amplified at low pH, where the proteins are less stable. Thermodynamic signatures and redox reactivity of the model Cys-bound hemes highlight the critical role of the protein scaffold and its dynamics in modulating redox-linked transitions between thiols and thiolates.

Cytochrome c (cyt c) is one of the most widely studied biomolecules, but not much is known about this protein from nematodes. Recombinant expression of Caenorhabditis elegans CYC-2.1 and CYC-2.2 allowed for detailed characterization of their structural features, redox properties, stabilities, and interactions with cardiolipin (CL)-containing liposomes. Using a variety of spectroscopic tools, we show that CYC-2.1 and CYC-2.2 adopt a globular α-helical fold with His/Met heme ligation. The longer CYC-2.2 has a lower thermodynamic stability than CYC-2.1 and lacks His residues to misligate to the heme in the protein’s denatured state. Both C. elegans proteins bind to CL-containing liposomes, and these interactions promote the proteins’ peroxidase activity but to a much greater degree for CYC-2.2. Dye-to-heme distance distributions from time-resolved fluorescence resonance energy transfer in bimane-labeled CYC-2.1 and CYC-2.2 revealed similar populations of extended and compact conformers for CL-bound proteins, suggesting that their distinct peroxidase activities in the presence of CL arise from differences in the local heme environments for the two polypeptide ensembles. Without inhibition from His misligation, a less stable and more prone to unfolding CYC-2.2 allows for better access of substrates to the heme and thus exhibits higher peroxidase activity. Similar features of the conformational ensembles of CYC-2.1 and CYC-2.2 to those of mammalian cyt c suggest that C. elegans proteins, particularly the former, could serve as useful models for examining the mechanism of cyt c–CL interactions in live organisms.

Using a collection of dye-labeled cytochrome c (cyt c) variants, we identify transformations of the heterogeneous cardiolipin (CL)-bound cyt c ensemble with added ATP. Distributions of dye-to-heme distances P(r) from time-resolved fluorescence resonance energy transfer show that ATP decreases the population of largely unfolded cyt c conformers, but its effects are distinct from those of a simple salt. The high peroxidase activity of CL-bound cyt c with added ATP suggests binding interactions that favor protein structures with the open heme pocket. Although ATP weakens cyt c–CL binding interactions, it also boosts the apoptosis-relevant peroxidase activity of CL-bound cyt c.

Interactions of cytochrome c (cyt c) with a unique mitochondrial glycerophospholipid cardiolipin (CL) are relevant for the protein’s function in oxidative phosphorylation and apoptosis. Binding to CL-containing membranes promotes cyt c unfolding and dramatically enhances the protein’s peroxidase activity, which is critical in early stages of apoptosis. We have employed a collection of seven dansyl variants of horse heart cyt c to probe the sequence of steps in this functional transformation. Kinetic measurements have unraveled four distinct processes during CL-induced cyt c unfolding: rapid protein binding to CL liposomes; rearrangements of protein substructures with small unfolding energies; partial insertion of the protein into the lipid bilayer; and extensive protein restructuring leading to “open” extended structures. While early rearrangements depend on a hierarchy of foldons in the native structure, the later process of large-scale unfolding is influenced by protein interactions with the membrane surface. The opening of the cyt c structure exposes the heme group, which enhances the protein’s peroxidase activity and also frees the C-terminal helix to aid in the translocation of the protein through CL membranes.

Interactions of cytochrome c (cyt c) with cardiolipin (CL) partially unfold the protein, activating its peroxidase function, a critical event in the execution of apoptosis. However, structural features of the altered protein species in the heterogeneous ensemble are difficult to probe with ensemble averaging. Analyses of the dye-to-heme distance distributions P(r) from time-resolved FRET (TR-FRET) have uncovered two distinct types of CL-bound cyt c conformations, extended and compact. We have combined TR-FRET, fluorescence correlation spectroscopy (FCS), and biolayer interferometry to develop a systematic understanding of the functional partitioning between the two conformations. The two subpopulations are in equilibrium with each other, with a submillisecond rate of conformational exchange reflecting the protein folding into a compact non-native state, as well as protein interactions with the lipid surface. Electrostatic interactions with the negatively charged lipid surface that correlate with physiologically relevant changes in CL concentrations strongly affect the kinetics of cyt c binding and conformational exchange. A predominantly peripheral binding mechanism, rather than deep protein insertion into the membrane, provides a rationale for the general denaturing effect of the CL surface and the large-scale protein unfolding. These findings closely relate to cyt c folding dynamics and suggest a general strategy for extending the time window in monitoring the kinetics of folding.

Ligand binding and substitution reactions are important for metalloprotein folding and function. The heme sensor of a methyl-accepting chemotaxis GSU0935 is a c-type cytochrome from the bacterium Geobacter sulfurreducens. The heme domain switches one of its axial ligands from H2O to a low-spin ligand, presumably Met, upon reduction. The study analyzes the stability and folding kinetics of the ferric domain. Guanidine hydrochloride denaturation yields the low-spin heme species arising from coordination of the ferric heme by non-native His residues. The population of the low-spin species further increases and then declines during protein refolding. Kinetics and mutational effects suggest that His54, from the N-terminal region of the domain, is the transient ligand to the heme. The capture and release of a non-native ligand within the compact partially-folded structures illustrates the flexibility of the heme environment in GSU0935, which may relate to the domain sensor function.

Interactions of cytochrome c (cyt c) with cardiolipin (CL) are important for both electron transfer and apoptotic functions of this protein. A sluggish peroxidase in its native state, when bound to CL, cyt c catalyzes CL peroxidation, which contributes to the protein apoptotic release. The heterogeneous CL-bound cyt c ensemble is difficult to characterize with traditional structural methods and ensemble-averaged probes. We have employed time-resolved FRET measurements to evaluate structural properties of the CL-bound protein in four dansyl (Dns)-labeled variants of horse heart cyt c. The Dns decay curves and extracted Dns-to-heme distance distributions P(r) reveal a conformational diversity of the CL-bound cyt c ensemble with distinct populations of the polypeptide structures that vary in their degree of protein unfolding. A fraction of the ensemble is substantially unfolded, with Dns-to-heme distances resembling those in the guanidine hydrochloride-denatured state. These largely open cyt c structures likely dominate the peroxidase activity of the CL-bound cyt c ensemble. Site variations in P(r) distributions uncover structural features of the CL-bound cyt c, rationalize previous findings, and implicate the prime role of electrostatic interactions, particularly with the protein C terminus, in the CL-induced unfolding.

A new thiol-specific reagent introduces a small bis(methylamino)terephthalic acid fluorophore into proteins. The noninvasive probe with distinct spectroscopic properties offers many advantages for protein labeling, purification, and mechanistic work promising to serve as a powerful tool in studies of protein folding and heme redox reactions.

A new thiol-specific reagent introduces a small bis(methylamino)terephthalic acid fluorophore into proteins. The noninvasive probe with distinct spectroscopic properties offers many advantages for protein labeling, purification, and mechanistic work promising to serve as a powerful tool in studies of protein folding and heme redox reactions.

Ligand binding and substitution reactions are important for metalloprotein folding and function. The heme sensor of a methyl-accepting chemotaxis GSU0935 is a c-type cytochrome from the bacterium Geobacter sulfurreducens. The heme domain switches one of its axial ligands from H2O to a low-spin ligand, presumably Met, upon reduction. The study analyzes the stability and folding kinetics of the ferric domain. Guanidine hydrochloride denaturation yields the low-spin heme species arising from coordination of the ferric heme by non-native His residues. The population of the low-spin species further increases and then declines during protein refolding. Kinetics and mutational effects suggest that His54, from the N-terminal region of the domain, is the transient ligand to the heme. The capture and release of a non-native ligand within the compact partially-folded structures illustrates the flexibility of the heme environment in GSU0935, which may relate to the domain sensor function.

Measurements of photoinduced Fe2+-to-Ru3+ electron transfer (ET), supported by theoretical analysis, demonstrate that mutations off the dominant ET pathways can strongly influence the redox reactivity of cytochrome c. The effects arise from the change in the protein dynamics mediated by the intraprotein hydrogen-bonding network.