1D NMR spectra of Fe²⁺ cyt c (pH 7.0, 22 °C) vs. urea concentration. Expanded plots are shown for the low-field region containing heme meso protons (left) and the high-field region containing methyl and M80 side-chain resonances (right).

¹⁵N-¹H HSQC spectra of native Fe²⁺ cyt c (red contours) and its metastable CO complex, M-CO_ms (green) recorded at 15 °C in 0.1 M sodium phosphate, pH 7.0. Cross peaks labeled in bold undergo especially large CO-induced chemical shift changes and can be assigned only in the unligated form.

Schematic free-energy diagrams for Fe²⁺ cyt c in the absence (black lines) and presence of CO (red lines) under native conditions (left panel) and moderately denaturing conditions (right panel). The free energies of the various states and relative barrier heights are consistent with the equilibrium and kinetic parameters in Table 3, respectively.

Absorption spectra (Soret and visible regions) of horse Fe²⁺ cyt c at pH 7.0 (0.1 M sodium phosphate), 20 °C, under native conditions (red; N-state), and under denaturing conditions (6.5 M GuHCl) in the absence (black; U-state) and presence of 1 mM CO (blue; U-CO state). The inset shows an expanded plot of the visible region (470–600 nm).

(a) Chemical shift changes associated with dissociation of the CO-ligand from M-CO_ms vs. residue number. ¹H/¹⁵N chemical shift changes were scaled as follows: Δδ(¹H, ¹⁵N) = sqrt[Δδ (¹H)² + (Δδ(¹⁵N)/10)²], where Δδ(¹H) is the CO-induced change in the NH proton chemical shift and Δδ(¹⁵N) the corresponding change in the ¹⁵N chemical shift. (b) Ribbon diagram of cyt c (pdb: 1hrc) indicating regions of cyt c undergoing structural changes upon binding of a CO ligand under native conditions. Side chains shown in ball-and-stick mode indicate residues undergoing large chemical shift changes (Δδ > 0.4 ppm; red) and intermediate changes (0.15 ppm ≤ Δδ ≤0.4 ppm; green).

(a) Normalized changes in ellipticity, θ at 225 nm and heme absorbance at selected wavelengths vs. GuHCl concentration for Fe²⁺ cyt c (pH 7.0, 20 °C) in the presence of 1 mM CO (green and blue symbols) and absence of ligand (red and yellow symbols). (b) Relative populations of the three states (N: red; M-CO: green; U-CO: blue) populated during unfolding of Fe²⁺ cyt c in the presence of CO, as predicted by equilibrium parameters in Table 1. The dashed line shows the population of the U-state accumulating during two-state unfolding in the absence of CO.

(a) Normalized peak intensities of selected resonances resolved by 1D NMR (Figure 4) vs. urea concentration in CO-saturated D₂O solution. Red and yellow symbols indicate resolved peaks assigned to the N-state. Green triangles indicate the intensity of the peak at ~9.6 ppm due to the δ-meso proton in the M-state. Blue circles indicate the normalized intensity of the peak at 10.05 ppm assigned to the α-meso proton in the U-state. (b) Normalized peak intensities for the N-state resonances of the M80 methyl and two meso protons vs. GuHCl concentration in the presence of 1 mM CO (colored symbols) and absence of ligand (gray symbols).

Our NMR evidence that M-CO_ms has a well ordered structure distinct from that of the native state indicates that a substantial conformational change is required to allow binding of a CO ligand under non-denaturing conditions. This conclusion is consistent with the well-known fact that CO does not directly bind the native state of Fe²⁺ cyt c, unless it is destabilized in the presence of moderate concentrations of denaturant or extreme pH values.12,58,59 Thus, we postulate that a native-like intermediate, M, exists even in the absence of CO. This hypothesis, which will be further justified below, leads to the following expanded scheme to describe the equilibrium between native, intermediate and unfolded forms of cyt c and cyt c-CO (Scheme 2):

(a) Absorbance changes at selected wavelengths in the Soret region associated with GuHCl-induced unfolding of Fe²⁺ cyt c (pH 7.0, 20 °C) in the presence of 1 atm (~1 mM) CO. The lines represent a global fit of a three-state unfolding mechanism (Scheme 1) to the combined absorbance data (transition curves between 380 and 430 nm every 0.5 nm). (b) Intrinsic Soret absorbance spectra of the N-, M-CO and U-CO states obtained by plotting the respective extinction coefficients (local fitting parameters) vs. wavelength.

(a) Rate constant of CO rebinding to unfolded Fe²⁺ cyt c (in 6 M GuHCl, 0.1 M sodium phosphate, pH 7.0) vs. CO concentration measured at 10 °C by laser flash photolysis (see text). (b) Arrhenius plot of the rate of CO recombination to unfolded Fe²⁺ cyt c measured by laser flash photolysis at variable temperature (5–30 °C) and a constant CO concentration of 135 µM. (c) Rate constant for heme absorbance changes upon addition of imidazole to CO-bound Fe²⁺ cyt c (in 6.4 M GuHCl, 0.1 M Tris-HCl, pH 8.0) vs. total imidazole concentration. The limiting value reached at high imidazole concentration represents the rate of CO dissociation.

(a) Absorbance spectra of Fe²⁺ cyt c in the Soret region as a function of total CO concentration ranging from 0 nM to 50 µM under unfolding conditions at 20 °C. The CO concentrations are color-coded as shown in the legend. The buffer contained 0.1 M Tris-HCl (pH 7.5), 6.5 M GuHCl, 3 mM dithionite. (b) CO binding curve (circles) monitored by absorbance at 413 nm as a function of total CO concentration. The solid line is a theoretical binding curve obtained by fitting the data based on Eq. 2.

(a) Arrhenius plot of the apparent rate of CO binding to Fe²⁺ cyt c at 2.5 M GuHCl (0.1 M sodium phosphate, pH 7.0, 1 mM CO) measured by manual mixing. (b) Logarithmic plot of the rate constant of thermally activated CO dissociation from the metastable CO complex (M-CO_ms) vs. GuHCl concentration (open squares). Also shown are the rate constants of unfolding of Fe²⁺ cyt c in the absence of CO (open circles) measured by absorbance-detected stopped-flow unfolding measurements at high GuHCl concentrations. The solid line represents the observable rate constant for the coupled CO-binding/unfolding process predicted on the basis of Scheme 2. The dashed lines indicate the elementary rate constants used for kinetic modeling.

According to Scheme 2, CO binding under non-denaturing conditions can be expressed in terms of the following reaction mechanism (Scheme 3) where the rate constants k_NM and k_MN describe the structural transition between the N and M-states, k_on ^M is the second-order CO binding rate to the M-state and k_off ^M represents the corresponding dissociation rate. We already know one of the rate constants in this scheme, namely that of the late folding event, k_MN = 8.3 × 10⁴ s⁻¹, which was measured by Pabit et al. 25 in their flash photolysis experiments starting from the metastable M-CO_ms state. Since the N ⬄ M equilibrium is strongly displaced towards N at low denaturant concentrations, we have k_MN ≫ k_NM. Judging from the magnitude of the CO binding rate to U (Table 2), we also expect that k_MN ≫ k_on ^M. Under these conditions (analogous to hydrogen exchange in the EX₂ limit), the observed rate for CO binding, k_obs, can be approximated as (1) where k_NM/k_MN is the equilibrium constant for the N ⬄ M pre-equilibrium, K_NM, and k_on ^M is the apparent rate for CO binding to the M-state at a given CO concentrations (e.g., 1 mM in a saturated solution at 1 atm). Since K_NM ≪ 1 under native conditions, Eq. 1 explains how the apparent rate of CO binding at low denaturant concentration can be very slow, even if the intrinsic CO binding rate to the M-state is relatively fast.

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 absorption spectra of horse Fe²⁺ cyt c in the folded and the 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 6^th iron coordination site (because of the covalent linkage of the heme via Cys14 and Cys17, the native His18 ligand remains bound to the 5^th coordination site under the denaturing conditions used here7). To measure the effect of CO on the unfolding equilibrium, we recorded absorbance spectra on a series of Fe²⁺ cyt c samples at different GuHCl concentrations equilibrated under 1 atm CO (~1 mM). Figure 2(a) shows the absorbance changes at selected wavelengths in the Soret region as a function of GuHCl concentration. At certain wavelengths (e.g., 410 nm) gradual changes in absorbance occur already at GuHCl concentrations between 1 and 2.7 M while a single steeper transition centered around 3.6 M GuHCl is observed at other wavelengths (most pronounced at 418.5 nm). The two transitions are especially well resolved in the curve monitored at 416.5 nm, which shows two resolved phases with opposite sign resulting in a bell-shaped curve. These observations provide clear evidence for the presence of an intermediate state with absorbance properties distinct from the equilibrium states populated at low (N) and high (U-CO) denaturant concentrations. The solid lines in Figure 2(a) were obtained by fitting a three-state mechanism (Scheme 1) to the transition curves measured at 0.5 nm intervals over the range from 380 to 430 nm, using global fitting procedures described by Latypov et al.48 The equation used to describe Scheme 1 for the global analysis involves a total of 8 parameters; the global parameters, C_m1, m₁, C_m2 and m₂ listed in Table 1 describe the two coupled denaturant-induced unfolding transitions, and the local parameters ε_N, ε_M-CO, ε_U-CO and s_U describe the wavelength-dependence of the data. A slope s_U = d(ε_U-CO)/dc was included to account for the effect of GuHCl on the absorbance of the fully unfolded CO complex. The spectral parameters, ε_N, ε_M-CO and ε_U-CO, plotted in Figure 2(b) vs. wavelength, represent the intrinsic absorption spectra of the three conformational states. ε_N is identical to the spectrum of the native state while ε_U-CO represents the spectrum of U-CO extrapolated to 0 M GuHCl, which is similar to the spectrum of U-CO measured at high GuHCl concentrations (cf. Figure 1). The Soret band of the M-CO state is more intense than both N and U-CO, and its absorption maximum (λ_max = 413.5 nm) falls in between those of N (414.5 nm) and U-CO (412.5 nm).