Folding Mechanism of Reduced Cytochrome c: Equilibrium and Kinetic Properties in the Presence of Carbon Monoxide

Abstract

Despite close structural similarity, the ferric and ferrous forms of cytochrome c (cyt c) differ greatly in terms of their ligand binding properties, stability, folding and dynamics. The reduced heme iron binds diatomic ligands such as CO only under destabilizing conditions that promote weakening or disruption of the native methionine-iron linkage. This makes CO a useful conformational probe for detecting partially structured states that cannot be observed in the absence of endogenous ligands. Heme absorbance, circular dichroism and NMR were used to characterize the denaturant-induced unfolding equilibrium of Fe 2+ cyt c in the presence and absence of CO. In addition to the native state (N), which does not bind CO, and the unfolded CO-complex (U-CO), a structurally distinct CObound form (M-CO) accumulates to high levels (~75% of the population) at intermediate guanidine hydrochloride concentrations. Comparison of the unfolding transition for different conformational probes reveals that M-CO is a compact state containing a native-like helical core and regions of local disorder in the segment containing the native Met80 ligand and adjacent loops. Kinetic measurements of CO binding and dissociation under native, partially denaturing and fully unfolded conditions indicate that a state, M, that is structurally analogous to M-CO is populated even in the absence of CO. The binding energy of the CO ligand lowers the free energy of this high-energy state to such an extent that it accumulates even under mildly denaturing equilibrium conditions. The thermodynamic and kinetic parameters obtained in this study provide a fully self-consistent description of the linked unfolding/CO-binding equilibria of reduced cyt c.

Investigations of the Low-Frequency Spectral Density of Cytochrome c upon Equilibrium Unfolding

“…In this paper we use UV-vis spectroscopy and a previously proposed procedure 30 to observe an intermediate state of unfolded cyt c in the range of 1.5 – 3 M GdHCl. Further investigations reveal that the intermediate state has little effect on the higher frequency RR spectra.…”

X-ray snapshots reveal conformational influence on active site ligation during metalloprotein folding

“…As suggested by Latypov and coworkers, CO-bound cyt c could assume multiple disordered conformations. 62 Such an ensemble of disordered states may explain the parallel folding processes observed in many of the previous timeresolved studies on CO-bound cyt c. 11,15,57 Aer photolysis, the subpopulations with an intact local structure assume the Fe-Met80 ligation state, and the ligation is protected throughout the folding process. On the other hand, the subpopulations without an intact local structure should adopt a ligation state with lowest energy solely due to metal-ligand bond strength, in which case the Fe-HisX ligations dominate.…”

Viscosity-dependent structural fluctuation of the M80-containing Ω-loop of horse ferrocytochrome c

“…The NCO-state is prepared from chemically denatured cyt c by lowering the denaturant concentration in the presence of carbon monoxide, which binds tightly to the heme iron and prevents formation of the native M80-heme (sulfur-iron) link [57,58,64]. Briefly, cyt c is unfolded in the presence of $6 M GdnHCl.…”

“…In this work, we have examined the influence of solvent viscosity on the structural fluctuation of presumably the M80-containing X-loop by measuring the rate of thermally-driven CO-dissociation from the natively folded carbonmonoxycytochrome c (NCO-state). Because of its smaller size and the content of the heme iron, carbonmonoxycytochrome c is a model in experimental protein dynamics studies [41,[57][58][59][60][61][62][63]. The CO-dissociation rate of NCOstate varies inversely with viscosity initially when the solvent viscosity is low (68.0 cP), but saturates at higher viscosity, indicating that the CO-dissociation from the NCO-state involves sequential stages that depend differently on solvent friction, i.e., solvent coupled and nonsolvent-coupled stages of the process [22,47].…”