Investigations of heme distortion,low-frequency vibrational excitations,and electron transfer in cytochrome c

Cytochrome (cyt) c is an important electron transfer protein. The ruffling deformation of its heme cofactor has been suggested to relate to its electron transfer rate. However, there is no direct experimental evidence demonstrating this correlation. In this work, we studied Pseudomonas aeruginosa cytochrome c₅₅₁ and its F7A mutant. These two proteins, although similar in their X-ray crystal structure, display a significant difference in their heme out-of-plane deformations, mainly along the ruffling coordinate. Resonance Raman and vibrational coherence measurements also indicate significant differences in ruffling-sensitive modes, particularly the low-frequency γ_a mode found between ∼50–60 cm⁻¹. This supports previous assignments of γ_a as having a large ruffling content. Measurement of the photoreduction kinetics finds an order of magnitude decrease of the photoreduction cross-section in the F7A mutant, which has nearly twice the ruffling deformation as the WT. Additional measurements on cytochrome c demonstrate that heme ruffling is correlated exponentially with the electron transfer rates and suggest that ruffling could play an important role in redox control. A major relaxation of heme ruffling in cytochrome c, upon binding to the mitochondrial membrane, is discussed in this context.Cytochrome (cyt) c is an important electron transfer protein that is involved in a variety of biological functions such as photosynthesis, respiration, and apoptosis (1). The heme group (Fe-protoporphyrin IX) is the functional center of cyt c. The heme iron is axially coordinated to His18 (proximal ligand) and Met80 (distal ligand) in its native solution state. The porphyrin ring is also covalently anchored to the protein by two thioether linkages with Cys-14 and Cys-17, which form a Cys-X-X-Cys-His (CXXCH) pentapeptide unit that is a unique feature shared by nearly all c-type hemes (1) [“XX” refers to other amino acids, e.g., Val and Ala, as in Pseudomonas aeruginosa (Pa) cyt c₅₅₁].The heme in cyt c has a geometry that is dominated by a large ruffling distortion, induced by both the protein fold and by the CXXCH motif (2, 3). Systematic analysis of X-ray crystal structures of heme proteins has shown that the proteins belonging to the same functional class share similar out-of-plane (OOP) heme distortions (4–6). These protein-induced OOP distortions are energetically unfavorable for the heme, and their evolutionary conservation implies that they have biological significance. Among them, doming and ruffling have been reasonably well characterized and correlated with protein functions. Doming is typically observed in oxygen storage or transport proteins such as hemoglobin (7, 8) and myoglobin (9). Moreover, the coupling of heme doming to the protein conformational substates has been shown to be functionally significant in a variety of heme protein systems (10–12). However, heme ruffling, which is the primary topic of this paper, is the dominant OOP deformation found in c-type cytochromes (4–6, 13) and nitrophorins (14–16), which are involved in electron and NO transport, respectively.As seen in Fig. 1, ruffling involves a pyrrole-ring twisting about the Fe–N bond. The ruffling distortion tilts the p_z orbitals of the porphyrin nitrogens away from the heme normal and increases overlap of the porphyrin a_2u and iron d_xy orbitals. It has been shown (17) via NMR experiments and density functional theory computation that, in the absence of a strong π-acceptor axial ligand (18), a ruffling deformation increases the Fe 3d_π-based electron density on the iron center, which makes the heme meso-carbon electron donation to the iron 3d_xy orbital less energetically favorable (17). Ruffling destabilizes all three occupied Fe 3d-based molecular orbitals and decreases the positive and negative spin density on the β-pyrrole and meso-carbon, respectively (17). Consequently, the electron transfer rate to the ferric heme is expected to decrease as a function of the ruffling deformation (17). In addition, when ruffling is considered in isolation, it decreases the reduction potential of ferric cyt c (19–22).Open in a separate windowFig. 1.Crystal structure and NSD analysis of hemes in ferric Pa cyt c₅₅₁ and its F7A mutant are compared with hh cyt c. The minus sign of displacement is defined only for doming and inverse doming to indicate the direction of Fe displacement (+, proximal; −, distal). The ruffling mode is shown at the lower left part of the figure and the arrows indicate the rotation of pyrrole rings with respect to Fe–N axis (dotted black lines).The CXXCH pentapeptide in cyt c may be critical to the ruffled structure and the function of cyt c (2, 3, 23). The CXXCH unit is thought to affect the heme reduction potential (1), and it can influence heme deformation through the covalent bonding of the thioether groups and by hydrogen bonding within the pentapeptide (2, 3). Furthermore, the CXXCH pentapeptide may have a biologically important role related to its proximity to the electron transfer partner binding site, as in the yeast cyt c peroxidase/cyt c complex (24). The local vibrational modes of heme in the 250–400 cm⁻¹ region have been shown to strongly mix with the vibrational modes of the CXXCH motif (23). This suggests that the heme–CXXCH vibrational dynamic couplings can play a role in electron transfer by coupling the vibrations of the heme directly to vibrations of the CXXCH unit at the protein–protein interface. This coupling could help to transduce thermal energy or alter the reorganization energy and the barrier for electron tunneling (23).Despite the great deal of work that has been done to investigate electron transfer and heme deformation in cyt c, no experiment has directly demonstrated a quantitative correlation between heme deformation and the electron transfer rate. Generally, the functionally important heme modes, such as doming and ruffling, are delocalized and involve many nuclei and lie in the low-frequency region below 200 cm⁻¹. Infrared and resonance Raman spectroscopy cannot reliably detect heme modes below ∼150 cm⁻¹ in the aqueous phase, owing to the strong absorbance, Rayleigh scattering, and quasi-elastic scattering of water (25). In contrast, impulsive stimulated Raman driven vibrational coherence, or vibrational coherence spectroscopy (VCS), makes it possible to extract vibrational modulations of the third-order polarization of the heme at very low frequency, which provides access to this relatively unexplored region.We have used this technique previously to investigate the low-frequency modes of a variety of heme proteins, using Soret band excitation (26–29). Unlike the higher frequency modes (>200 cm⁻¹), the low-frequency modes (which have weaker force constants) are more easily distorted from equilibrium by the protein surroundings. These modes are activated in VCS when the protein induces symmetry-breaking nonplanar heme distortions (29). In addition, these modes take on a special functional significance because of their thermal accessibility. The low-frequency coherence spectra offer a unique window into how the surrounding protein environment can alter these important thermally active heme modes.In this work, we studied Pa cyt c₅₅₁ and its F7A mutant using absorption spectroscopy, resonance Raman spectroscopy, and VCS. Pa cyt c₅₅₁ and its F7A mutant have very similar crystal structures, but the mutant has a more ruffled heme geometry than the WT. The investigations of this very similar pair of proteins revealed a clear difference between their resonance Raman and VCS spectra, reflecting the different degree of heme ruffling deformation. These observations support the previous assignment that γ_a (45∼60 cm⁻¹) is a mode with major ruffling content in the c-type heme of cyt c. We also investigated the photoreduction kinetics of the two cyt c₅₅₁ proteins as well as horse heart cyt c (hh cyt c hereafter). The photoreduction cross-section determined for WT cyt c₅₅₁ is an order of magnitude larger than for the more ruffled F7A mutant and approximately two orders of magnitude larger than hh cyt c. Although the details of photoreduction in heme proteins are not fully understood (30–32), these measurements provide direct quantitative evidence that correlate dramatic increases in the photoinduced electron transfer rate with only approximately a factor of two decrease in the ruffling distortion.