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

Abstract

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.

Engineered holocytochrome c synthases that biosynthesize new cytochromes c

“…As expected, the human WT cyt c shows a potential of +252 mV vs. standard hydrogen. The redox potential for cyt c M81A (−19 mV) was over 270-mV more negative than WT cyt c. A similar potential was determined previously for yeast iso-1-cyt c M80A (−80 mV) (20). The potential of cyt c M81H (−69 mV) suggests a stabilization of the oxidized cytochrome, similar to the chemically generated horse heart bis-His cyt c variant (+41 mV) (11); such a change is often observed when substituting a stronger histidine ligand for methionine.…”

“…In addition, pronounced peaks are present at 2.08 and 1.97 in human cyt c M81A. These are also observed in the yeast iso-cyt c M81A variant, albeit with weaker intensity (20,21). Human cyt c M81H exhibits g values at 2.91, 2.29, and 1.58, very similar to a cytochrome b562 M7H with bisHis ligands to b heme and bis-imidazole-ligated hemin (23) ( Table S3).…”

Ligation and Reactivity of Methionine-Oxidized Cytochrome c

“…The dynamics of the heme coordination loop have been thoroughly characterized for yeast iso -1 cyt c . 40 Further, many of its previously characterized mutants, including M80K # from our recent work, 21 facilitate analysis of the ligation to the heme iron.…”

“…Studies of cyt c mutants have revealed that His, Lys, and hydroxide ligands give rise to very similar positions of the Soret band, blue-shifted by a few nm from its position at 409 nm for the Met-ligated WT*. 17,21,43–45 The far-UV and near-UV CD spectra of Met-SO and native cyt c (Figure S2) suggest only minor perturbations in the secondary and tertiary structure by the modification. Thus the ligation by His26, 33, or 39 residues, which are remote from the heme coordination loop, is unlikely.…”

“…14,27,61–63 Acetylation of the N -terminus of MP8 diminishes formation of the peptide dimers. Using AcMP8, we have previously quantified the binding constants to the ferric 21 and ferrous 61 heme iron for Lys. To enable the comparison of Lys and Met-SO, we now wanted to extend these measurements to Met-SO.…”