Probing the Bottom of a Folding Funnel Using Conformationally Gated Electron Transfer Reactions

Abstract

The effect of global stability on the kinetics of interconversion between the native (N) and a compact, partially unfolded form (I) of iso-1-cytochrome c stabilized by His73-heme ligation is investigated using a novel conformationally gated ET method. For the K73H variant and the 2-fold less stable AcH73 variant, the N and I conformers are of nearly equal stability at pH 7.5. The pH jump kinetic data yield kobs = kNI + kIN of 35-40 s-1 at final pH values from 6 to 8 for the AcH73 variant, about 3-fold faster than for the more stable K73H variant. Gated ET measurements give kNI = 28 s-1 and kIN = 13 s-1 for the AcH73 variant, 10- and 2-fold greater than that for the more stable K73H variant. Thus, funneled landscapes have evolved such that loss of global stability lowers barriers at the bottom of a folding funnel, still allowing for efficient folding.

Conformational Toggling of Yeast Iso-1-Cytochrome c in the Oxidized and Reduced States

“…However, the CD spectrum of oxidized P71H variant was much different from that of the oxidized native cyt c (Figure S4-C), but consistent with that of oxidized yeast iso-1 cyt c F82H mutant [10]–[13], [19], especially in the negative absorption at 418 nm, which is characteristic of the interaction between Met80 and heme iron [29]–[31]. The almost identical CD spectra of reduced P71H mutant with the reduced native cyt c were seen in Figures S4-C and S4-D, consistent with the results from its UV spectrum above.…”

“…It seems that the histidine introduced at position 71 likely acted as a strong sixth axial ligand of heme iron in the oxidized state at pH 7.0, while in the reduced state of the P71H variant at pH 7.0, the axial iron ligands switched to His18 and Met80, as did in the native cyt c . This kind of axial ligand conformation switching of cyt c was claimed in the F82H and K79H mutants of cyt c [10]–[13], [19], where His82 and His79 were thought to be the histidine on the distal side of the heme in the oxidized state of yeast iso-1 cyt c . However, there was no solid structural evidence to support this kind of conformational toggling.…”

“…Methionine and histidine are the invariable axial ligands of native mitochondrial cyt c , other variants have these ligands substituted or deleted [1], [4]. Several factors that were closely associated with the changes in axial coordination had been reported as pH linked conformational changes (the native form versus the alkaline form) [5]–[7], redox “switched” changes (folded versus unfolded form) [8]–[13], and the refolding of cyt c [14]–[18]. These coordination changes and rearrangements were suggested as a mechanism for converting redox energy into conformational energy, or as a switch from an intermediate His/His coordinated form to the native Met/His form [15]–[19].…”

Effect of Antimicrobial Preservatives on Partial Protein Unfolding and Aggregation

“…When the local region around Met80 unfolds, this absorption band is absent because of the breakage of the Met80 to heme bond. The identification of the Ω–loop containing Met80 as being a least stable region, or weakest link in the protein, came from earlier residue-resolved hydrogen exchange (HX) experiments measured using two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy 28–31 , protein unfolding monitored in response to various stresses 32–34 , and from studies on ultrafast protein dynamics 35 . Our earlier computational analysis predicts that the six residues around Met80 acts as an aggregation “hot-spot” whose unfolding may lead to Cyt c aggregation 36 , and experimental results confirm this hypothesis 36 .…”

Structural Analysis of Diheme Cytochrome c by Hydrogen–Deuterium Exchange Mass Spectrometry and Homology Modeling

“…16−23 Although some comparative X-ray crystallographic studies of tuna heart cytochromes c show little or no difference between the backbone structures, 24 almost all solution-based studies show a clear conformational change between the two redox states. 25−27 Specifically, in the case of horse heart cytochrome c , the radius of the oxidized state is significantly larger than that of the reduced state.…”