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.

Cytochrome c, a mitochondrial electron transfer protein containing a hexacoordinated heme, is involved in other physiologically relevant events, such as the triggering of apoptosis, and the activation of a peroxidatic activity. The latter occurs secondary to interactions with cardiolipin and/or post-translational modifications, including tyrosine nitration by peroxynitrite and other nitric oxide-derived oxidants. The gain of peroxidatic activity in nitrated cytochrome c has been related to a heme site transition in the physiological pH region, which normally occurs at alkaline pH in the native protein. Herein, we report a spectroscopic characterization of two nitrated variants of horse heart cytochrome c by using optical spectroscopy studies and NMR. Highly pure nitrated cytochrome c species modified at solvent-exposed Tyr-74 or Tyr-97 were generated after treatment with a flux of peroxynitrite, separated, purified by preparative high pressure liquid chromatography, and characterized by mass spectrometry-based peptide mapping. It is shown that nitration of Tyr-74 elicits an early alkaline transition with a pK a ‫؍‬ 7.2, resulting in the displacement of the sixth and axial iron ligand Met-80 and replacement by a weaker Lys ligand to yield an alternative low spin conformation. Based on the study of site-specific Tyr to Phe mutants in the four conserved Tyr residues, we also show that this transition is not due to deprotonation of nitro-Tyr-74, but instead we propose a destabilizing steric effect of the nitro group in the mobile ⍀-loop of cytochrome c, which is transmitted to the iron center via the nearby Tyr-67. The key role of Tyr-67 in promoting the transition through interactions with Met-80 was further substantiated in the Y67F mutant. These results therefore provide new insights into how a remote post-translational modification in cytochrome c such as tyrosine nitration triggers profound structural changes in the heme ligation and microenvironment and impacts in protein function.

Cytochrome c (cyt c) is a cationic hemoprotein of ∼100 amino acid residues that exhibits exceptional functional versatility. While its primary function is electron transfer in the respiratory chain, cyt c is also recognized as a key component of the intrinsic apoptotic pathway, the mitochondrial oxidative protein folding machinery, and presumably as a redox sensor in the cytosol, along with other reported functions. Transition to alternative conformations and gain-of-peroxidase activity are thought to further enable the multiple functions of cyt c and its translocation across cellular compartments. In vitro, direct interactions of cyt c with cardiolipin, post-translational modifications such as tyrosine nitration, phosphorylation, methionine sulfoxidation, mutations, and even fine changes in electrical fields lead to a variety of conformational states that may be of biological relevance. The identification of these alternative conformations and the elucidation of their functions in vivo continue to be a major challenge. Here, we unify the knowledge of the structural flexibility of cyt c that supports functional moonlighting and review biochemical and immunochemical evidence confirming that cyt c undergoes conformational changes during normal and altered cellular homeostasis.