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A Compact Structure of Cytochrome c Trapped in a Lysine-Ligated State: Loop Refolding and Functional Implications of a Conformational Switch

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Department of Biochemistry, Geisel School of Medicine, Hanover, New Hampshire 03755, United States
Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755, United States
§ Department of Chemistry, Berry College, Mount Berry, Georgia 30149, United States
Cite this: J. Am. Chem. Soc. 2015, 137, 26, 8435–8449
Publication Date (Web):June 3, 2015
Copyright © 2015 American Chemical Society

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    Abstract Image

    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.

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    Figures showing charge-transfer absorption bands for T78C/K79G, T78C/K79G/M80L, and WT*, effects of CL liposomes on the absorption spectra of T78C/K79G, spectroelectrochemistry curves for T78C/K79G, T78C/K79G/M80L, and WT*, and 1D 1H and 2D 1H NOESY NMR spectra of ferrous T78C/K79G; results of thermal denaturation for ferric and ferrous T78C/K79G, T78C/K79G/M80L, and WT*; pH effects on the T78C/K79G absorption spectra; 1D 1H NMR spectra of ferric WT*, T78C/K79G, and myoglobin; electron density for the heme in the crystal structure, arrangement of protomers, and dimerization contacts; heme iron coordination geometry in T78C/K79G and other proteins with Lys ligation at the heme iron; analysis of the dimer content in solution; binding of AcLys to AcMP8; spectra of ferric and ferrous K73A/K79G/M80K compared to that of WT*; pH effects on the absorption spectra of ferric K73A/K79G/M80K; voltammograms of the crystal drop sample; changes in the absorption spectra of T78C/K79G after addition of PEG300; comparison of structures with Lys73 coordinated to the heme iron solved by X-ray crystallography and NMR; comparison of the T78C/K79G dimer to the dimeric structure (after alcohol treatment) of horse heart cyt c; environment of the heme propionate HP6; analysis of the shift in the 50s helix; reversibility of thermal denaturation; and tables listing results of thermal denaturation; EPR parameters; and pKa values for the studied variants; data collection and refinement statistics; and comparison of thermodynamic parameters of different variants. Coordinates and structure factors were deposited in the Protein Data Bank, with accession code 4Q5P. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.5b01493.

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