Journal of Molecular Biology
Volume 383, Issue 2, 7 November 2008, Pages 437-453
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Folding Mechanism of Reduced Cytochrome c: Equilibrium and Kinetic Properties in the Presence of Carbon Monoxide

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Abstract

Despite close structural similarity, the ferric and ferrous forms of cytochrome 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 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 ferrocytochrome c in the presence and in the 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 CO-bound form (M-CO) accumulates to high levels (∼ 75% of the population) at intermediate guanidine HCl concentrations. Comparison of the unfolding transitions 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 cytochrome c.

Introduction

Metalloproteins often depend on their cofactor not only for function but also for efficient folding and stabilization of their native structure. For example, in the absence of heme, myoglobin folds into a marginally stable state lacking some of the native α-helices and tertiary interactions,1 and cytochrome b562 assumes a dynamic molten-globule-like conformation.2 An even more extreme case is cytochrome c (cyt c), which is initially synthesized as a largely disordered apoprotein3, 4 and folds into a stable globular structure only after covalent attachment of the heme.5, 6 These observations are not inconsistent with the notion that the native structure of a protein is encoded in its sequence, but suggest that the cofactor carries some of the information defining the native structure.

It has long been known that the heme and its axial ligands have a profound influence on the folding process of horse cyt c.7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 Similar findings have been reported for c-type cytochromes from other species, including yeast and photosynthetic bacteria,18, 19 but we focus here on horse cyt c, which has been studied especially thoroughly. Under typical denaturing conditions [e.g., 6 M guanidine HCl (GuHCl), pH > 4], one of the two axial heme ligands, the imidazole nitrogen of His18, remains bound to the heme iron due to the fact that the adjacent Cys17 is covalently bound to the heme. However, coordination of the second axial ligand, the sulfur of Met80, is inherently less stable and readily dissociates under partly or fully denaturing conditions. In unfolded cyt c, the vacant heme coordination site can bind alternative ligands, including extraneous ligands [such as imidazole or, in the case of the reduced (Fe2 +) form, carbon monoxide], as well as intramolecular ligands (such as His, Lys, or the amino terminus in their deprotonated states). Detailed studies of such ligand exchange reactions in both iron oxidation states have provided a rich source of information on the conformational propensities and dynamics of the denatured state of cyt c.12, 20, 21, 22, 23, 24, 25, 26, 27 The predominant sixth iron ligand in the unfolded state of oxidized (Fe3 +) cyt c is His33,28 which can become trapped during refolding and leads to accumulation of relatively long-lived (∼ 100 ms) intermediate states that feature both native-like and nonnative structural features.10, 13, 14

Although the structures of the oxidized and reduced forms of mitochondrial cyt c—as determined by X-ray crystallography29, 30, 31 or NMR32, 33, 34, 35—are very similar, the two forms differ greatly in terms of stability, dynamics, and folding kinetics.12, 36, 37, 38, 39, 40, 41 The dramatic stabilization of the protein upon reduction of the heme is not fully understood, but appears to be due to a combination of electrostatic effects (the reduced heme is electrically neutral, while the oxidized heme carries a net charge of + 1), differential affinity of the heme iron for axial ligands between the native and the unfolded conformations in both oxidation states, and dynamic/entropic contributions. The fact that only nonnative forms of ferrocytochrome c (Fe2 + cyt c) bind a CO ligand with high affinity to form a photolabile ligand complex has opened unique opportunities for manipulating the conformational transitions of the protein and for probing its dynamics on the microsecond-to-millisecond time scale.12, 20, 21, 23, 25, 27, 42, 43 In their initial equilibrium characterization of Fe2 + cyt c in the presence of CO, Jones et al. had already noticed that this system does not undergo a simple two-state unfolding transition.12 When observing the GuHCl-induced unfolding transition for Fe2 + cyt c at 40 °C using tryptophan fluorescence, they measured a midpoint concentration Cm = 3.7 M (m = 2.6 kcal mol 1 M 1) in the presence of CO (1 atm, corresponding to ∼ 1 mM CO in solution) and a midpoint concentration Cm = 5.1 M (m = 3.6 kcal mol 1 M 1) in the absence of CO. These parameters indicate that addition of CO results in a nearly 9-kcal mol 1 decrease in the stability of native Fe2 + cyt c. A similar unfolding transition was observed using far-UV CD spectroscopy. However, changes in heme absorbance indicative of CO binding had already been observed between 1 and 3 M GuHCl, where the fluorescence and far-UV CD signals remain at their native levels.12 This is a clear indication that the unfolding transition in the presence of CO cannot be adequately described in terms of a two-state mechanism.

Despite this earlier evidence for noncooperative behavior, Bhuyan and colleagues concluded that reduced cyt c undergoes a two-state unfolding transition, both in the presence and in the absence of CO, based on their equilibrium and stopped-flow studies of folding and unfolding.40, 41, 44, 45 In order to resolve this controversy, we further characterized the unfolding equilibrium of Fe2 + cyt c in the presence and in the absence of CO using optical techniques and NMR. The results provide clear evidence that, in addition to the native state (N), which does not bind CO, and to the unfolded CO complex (U-CO), a structurally distinct CO-bound form (M-CO) accumulates at intermediate denaturant concentrations. Based on its optical and NMR properties, M-CO is a folded state with 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 and denaturing conditions confirm our hypothesis that a state M, which is structurally analogous to M-CO, is populated even in the absence of CO. Even though M does not accumulate to detectable levels at equilibrium, its presence can limit the rate constant of unfolding, giving rise to a downward curvature in the log(rate)-versus-[denaturant] plot at high denaturant concentration, similar to that previously observed for oxidized c-type cytochromes.13, 18, 46 The binding energy of the CO ligand lowers the free energy of this transient intermediate 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.

Section snippets

Effect of CO binding on absorbance-detected and CD-detected unfolding transition

The changes in heme coordination that accompany unfolding and CO binding give rise to major changes in the heme absorption spectrum of cyt c.12, 41, 47 Figure 1 shows the absorption spectra of horse Fe2 + cyt c in the folded and GuHCl–unfolded forms. Addition of CO to unfolded cyt c (U) results in a large increase in the extinction coefficient for Soret absorption, consistent with the formation of a hexacoordinate low-spin complex (U-CO), with CO displacing the native Met80 sulfur at the sixth

Conclusions

The biological activity of cyt c as a mitochondrial electron transport shuttle critically depends on maintaining the native His/Met heme coordination,68 and binding of alternative ligands is strongly disfavored under physiological conditions. However, CO and other exogenous ligands have such a strong affinity for binding 5-coordinate ferrous heme that they can displace the native methionine ligand under partially denaturing conditions. Our equilibrium and kinetic analyses of CO binding to

Materials and Methods

Horse heart cyt c from Sigma-Aldrich Co. (St. Louis, MO) was used without further purification. GuHCl and urea were of ultrapure grade (MP Biomedicals, Solon, OH). All the other chemicals were of reagent grade. GuHCl concentration was determined by refractive index measurements using a Reichert–Jung refractometer (Leica, Bannockburn, IL). The concentration of oxidized cyt c was determined spectrophotometrically using an extinction coefficient of 1.06 × 105 M 1cm 1 at 410 nm. Protein solutions

Acknowledgements

The work was supported by National Institute of Health grants R01 GM 056250 and CA06927, and an appropriation from the Commonwealth of Pennsylvania to the Institute for Cancer Research. We thank the Spectroscopy Support Facility at Fox Chase Cancer Center for maintaining the NMR and optical spectrometers, and the Regional Laser and Biotechnology Laboratory for access to nanosecond laser flash photolysis.

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      Furthermore, among chaotropic anions, the m-value was found to be most negative for perchlorate and least negative for bromide. Few earlier studies revealed that the CO association to Ferrocyt c (Fe2+- M80 + CO → Fe2+–CO + M80) is not a global probe of dynamics but rather it is a low-frequency local motions that control structural fluctuation of the M80-containing Ω-loop of protein [38, 52, 54–56]. Thus, the anion modulation of rate of CO association with Ferrocyt c reveals the way by which the structural fluctuation of Ω-loop act in responses to anion content in the reaction medium.

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    Present addresses: R. F. Latypov, Amgen, Inc., Seattle, WA 98119, USA; K. Maki, Department of Physics, Graduate School of Science, Nagoya University, Aichi 464-8602, Japan; S. D. Luck, DuPont Agricultural Biotechnology, Newark, DE 19714, USA.

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