Protein-heme interaction in hemoglobin: evidence from Raman difference spectroscopy.

Raman difference spectroscopy measurements on native and chemically modified human deoxyhemoglobins stabilized in either the R or the T quaternary structure revealed frequency differences in the oxidation state marker lines. The differences indicate that the R structure has an effective increase in the electron density of the antibonding pi* orbitals of the porphyrin rings. This increase is explained by a charge transfer interaction between donor orbitals and the pi* orbitals of the porphyrins. The relative amount of charge transferred, which is inferred from the Raman difference measurements, correlates with some but not all factors that influence the energetics of the quaternary structure equilibrium. In addition, the free energy of cooperativity for a variety of ligated proteins follows the same order as that of the degree of charge depletion of the pi* orbitals upon ligation as determined from the frequency of a Raman mode. The proposed electronic interaction between the protein and heme could result in energies large enough to provide a significant contribution to the energetics of hemoglobin cooperativity.     

Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria

In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H₂O₂ levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H₂O₂ because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1’s heme is labilized by hyperoxidation of the protein during the burst in H₂O₂ production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H₂O₂ in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H₂O₂ via a previously unidentified mechanism involving H₂O₂-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H₂O₂ detoxification.Cytochrome c peroxidase (Ccp1) is a monomeric nuclear encoded protein with a 68-residue N-terminal mitochondrial targeting sequence (1). This presequence crosses the inner mitochondrial membrane and is cleaved by matrix proteases (2, 3). Mature heme-loaded Ccp1 is found in the mitochondrial intermembrane space (IMS) in exponentially growing yeast (2, 3) but the point of insertion of its single b-type heme is unknown. Under strict anaerobic conditions, Ccp1 is present in mitochondria as the heme-free form or apoform (4). Once cells are exposed to O₂ and heme biosynthesis is turned on, apoCcp1 converts rapidly to the mature holoenzyme by noncovalently binding heme (5).It is well established that mature Ccp1 functions as an efficient H₂O₂ scavenger in vitro (6). Its catalytic cycle involves the reaction of ferric Ccp1 with H₂O₂ (Eq. 1) to form compound I (CpdI) with a ferryl (Fe^IV) heme and a cationic indole radical localized on Trp191 (W191^+•). CpdI is one-electron reduced by the ferrous heme of cytochrome c (Cyc1) to compound II (CpdII) with ferryl heme (Eq. 2), and electron donation by a second ferrous Cyc1 returns CpdII to the resting Ccp1^III form (Eq. 3):Ccp1^III + H₂O₂ → CpdI(Fe^IV, W191^+?) + H₂O[1]CpdI(Fe^IV, W191^+?) + Cyc1^II → CpdII(Fe^IV) + Cyc1^III[2]CpdII(Fe^IV) + Cyc1^II → Ccp1^III + Cyc1^III + H₂O.[3]Because Ccp1 production is not under O₂/heme control (4, 5), CCP activity is assumed to be the frontline defense in the mitochondria, a major source of reactive oxygen species (ROS) in respiring cells (7). Contrary to the time-honored assumption that Ccp1 catalytically consumes the H₂O₂ produced during aerobic respiration (8), recent studies in our group reveal that the peroxidase behaves more like a mitochondrial H₂O₂ sensor than a catalytic H₂O₂ detoxifier (9–11). Notably, Ccp1 competes with complex IV for reducing equivalents from Cyc1, which shuttles electrons from complex III (ubiquinol cytochrome c reductase) to complex IV (cytochrome c oxidase) in the electron transport chain (12).Because CCP activity in the IMS siphons electrons from energy production, an H₂O₂ sensor role for Ccp1 should be energetically more favorable for the cell. Key evidence for a noncatalytic role for Ccp1 in H₂O₂ removal is that the isogenic strain producing the catalytically inactive Ccp1^W191F protein accumulates less H₂O₂ than wild-type cells (10). In fact, this mutant strain exhibits approximately threefold higher catalase A (Cta1) activity than wild-type cells (10) whereas CCP1 deletion results in a strain (ccp1Δ) with negligible Cta1 activity and high H₂O₂ levels (5). Unlike Cta1, which is the peroxisomal and mitochondrial catalase isoform in yeast (13), the cytosolic catalase Ctt1 (14) exhibits comparable activity in the wild-type, Ccp1^W191F, and ccp1Δ strains (10). Given that both Ccp1 and Cta1 are targeted to mitochondria, we hypothesized that Ccp1 may transfer its heme to apoCta1 in respiring cells.Cta1 is nuclear encoded with embedded mitochondrial and peroxisomal targeting sequences (15). Like Ccp1, each monomer noncovalently binds a b-type heme and mature Cta1 is active as a homotetramer. Synthesis of the Cta1 monomer is under O₂/heme control such that the apoenzyme begins to accumulate only during the logarithmic phase of aerobic growth (16). Hence, its O₂/heme independent production (4, 5) allows apoCcp1 to acquire heme while cells are synthesizing apoCta1. This, combined with our observation that Cta1 activity increases in respiring cells producing Ccp1 or Ccp1^W191F but not in ccp1Δ cells (10), led us to speculate that respiration-derived H₂O₂ triggers heme donation from Ccp1 to apoCta1 within mitochondria.What experimental evidence would support heme donation by Ccp1? It has been demonstrated that mutation of the proximal heme Fe ligand, His175, to a residue with weak or no Fe-coordinating ability produces Ccp1 variants (H175P, H175L, H175R, and H175M) that undergo mitochondrial processing but do not accumulate in isolated yeast mitochondria (17). Presumably, reduced heme affinity allows the Ccp1 variants to unfold and cross the outer mitochondrial membrane (17). Hence, we argued that if wild-type Ccp1 donated its heme, the apoprotein would likewise exit mitochondria. Consequently, we examine here age-dependent Ccp1–green fluorescent protein (Ccp1-GFP) localization in live cells chromosomally expressing Ccp1 C-terminally fused to GFP as well as the distribution of wild-type Ccp1 between subcellular fractions. Because weakening or removal of the proximal Fe ligand on His175 mutation reduces heme affinity (17), His175 oxidation in wild-type Ccp1 should have a similar effect, which we investigate here. We further speculated that in the absence of apoCta1 as an acceptor for its heme, more Ccp1 would remain trapped in the IMS so we compare mitochondrial Ccp1 levels in wild-type and cta1∆ cells. Our combined results support triggering of heme donation from Ccp1 to apoCta1 by respiration-derived H₂O₂. Such H₂O₂-activated heme transfer between proteins has not been reported to date and its implications in H₂O₂ signaling are discussed.     

Definition of the catalytic site of cytochrome c oxidase: specific ligands of heme a and the heme a3-CuB center.

The three-subunit aa3-type cytochrome c oxidase (EC 1.9.3.1) of Rhodobacter sphaeroides is structurally and functionally homologous to the more complex mitochondrial oxidase. The largest subunit, subunit I, is highly conserved and predicted to contain 12 transmembrane segments that provide all the ligands for three of the four metal centers: heme a, heme a3, and CuB. A variety of spectroscopic techniques identify these ligands as histidines. We have used site-directed mutagenesis to change all the conserved histidines within subunit I of cytochrome c oxidase from Rb. sphaeroides. Analysis of the membrane-bound and purified mutant proteins by optical absorption and resonance Raman spectroscopy indicates that His-102 and His-421 are the ligands of heme a, while His-284, His-333, His-334, and His-419 ligate the heme a3-CuB center. To satisfy this ligation assignment, helices II, VI, VII, and X, which contain these histidine residues, must be in close proximity. These data provide empirical evidence regarding the three-dimensional protein structure at the catalytic core of cytochrome c oxidase.     

Erythrocyte membranes undergo cooperative, pH-sensitive state transitions in the physiological temperature range: evidence from Raman spectroscopy.

We have examined the Raman scattering from erythrocyte ghosts at 2700 to 3000 cm-1 (CH-stretching region). Plots of the intensity (I) of the 2930 cm-1 band relative to the intensity of the thermally stable 2850 cm-1 band, i.e., the [I2930/I2850] ratio, as a function of temperature reveal a sharp discontinuity, which at pH 7.4 has a lower limit of 38 degrees and is irreversible above 42 degrees. [I2930/I2850] is stable between pH 7.0 and pH 7.4, but increases or decreases sharply below pH 7.0 or above pH 7.5, respectively. Reduction of pH to 6.5 lowers the transition temperature by about 16 degrees, and a shift to pH 6.0 drops the transition range to 0 to 7 degrees. The above effects of temperature and pH on Raman scattering closely correspond to those detected by studies on the interaction of membrane protein fluorophores and lipid-soluble fluorescence quenchers [Bieri, V. and Wallach, D.F.H. (1975) Biochim. Biophys. Acta 406, 415-423]. Taken together, these results suggest that the transitions represent concerted process, involving hydrophobic amino acid residues and lipid chains at apolar protein-lipid boundaries.     

Spectroscopic analysis of the cytochrome c oxidase-cytochrome c complex: circular dichroism and magnetic circular dichroism measurements reveal change of cytochrome c heme geometry imposed by complex formation.

Binding of cytochrome c to cytochrome c oxidase induces a conformational change in both proteins as well as a change of the electronic structure of the heme of cytochrome c, indicating an altered heme c-protein interaction. This follows from the observation that the induced circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of the oxidase-cytochrome c complex in the Soret region differ from the summed spectra of oxidase plus cytochrome c. Spectral changes occur in the complex composed of either the two ferric or the two ferrous hemoproteins. The difference CD and MCD signals saturate at a ratio of 1 heme c per heme aa3. The difference spectra are specific to the cognate complex. The results are interpreted to reflect a direct relationship between the recognition/binding step and the electron-transfer reaction. The conformational rearrangement induced in cytochrome c by cytochrome c oxidase consists of a structural rearrangement of the heme environment and possibly a change of the geometry of the heme iron-methionine-80 sulfur axial bond. This rearrangement may decrease the reorganizational free energy of electron transfer by adjusting the heme c geometry to a state between that of ferri- and ferrocytochrome c.     

From the Cover: CcsBA is a cytochrome c synthetase that also functions in heme transport

Little is known about trafficking of heme from its sites of synthesis to sites of heme-protein assembly. We describe an integral membrane protein that allows trapping of endogenous heme to elucidate trafficking mechanisms. We show that CcsBA, a representative of a superfamily of integral membrane proteins involved in cytochrome c biosynthesis, exports and protects heme from oxidation. CcsBA has 10 transmembrane domains (TMDs) and reconstitutes cytochrome c synthesis in the Escherichia coli periplasm; thus, CcsBA is a cytochrome c synthetase. Purified CcsBA contains heme in an “external heme binding domain” for which two external histidines are shown to serve as axial ligands that protect the heme iron from oxidation. This is likely the active site of the synthetase. Furthermore, two conserved histidines in TMDs are required for heme to travel to the external heme binding domain. Remarkably, the function of CcsBA with mutations in these TMD histidines is corrected by exogenous imidazole, a result analogous to correction of heme binding by myoglobin when its proximal histidine is mutated. These data suggest that CcsBA has a heme binding site within the bilayer and that CcsBA is a heme channel.     

The heme groups of cytochrome o from Escherichia coli.

Cytochrome o, one of the two terminal ubiquinol oxidases of Escherichia coli, is structurally and functionally related to cytochrome c oxidase of mitochondria and some bacteria. It has two heme groups, one of which binds CO and forms a binuclear oxygen reaction center with copper. The other heme is unreactive toward ligands, exhibits strong interactions with the binuclear center, and is mainly responsible for the reduced-minus-oxidized alpha band. Protoheme has been thought to be the prosthetic group of b-type cytochromes, including cytochrome o. However, the hemes of cytochrome o are of a different kind, for which we propose the name heme O. Its pyridine hemochrome spectrum is blue-shifted by 4 nm relative to that of protoheme, and chromatographic behavior showed that it is much more hydrophobic than protoheme. Fast atom bombardment mass spectrometry yielded a molecular mass of 839 Da. Heme O is proposed to be a heme A-like molecule, containing a 17-carbon hydroxyethylfarnesyl side chain, but with a methyl residue replacing the formyl group.     

Conversion of a c type cytochrome to a b type that spontaneously forms in vitro from apo protein and heme: implications for c type cytochrome biogenesis and folding

Cytochrome c(552) from Hydrogenobacter thermophilus, a thermophilic bacterium, has been converted into a b type cytochrome, after mutagenesis of both heme-binding cysteines to alanine and expression in the cytoplasm of Escherichia coli. The b type variant is less stable, with the guanidine hydrochloride unfolding midpoint occurring at a concentration 2 M lower than for the wild-type protein. The reduction potential is 75 mV lower than that of the recombinant wild-type protein. The heme can be removed from the b type variant, thus generating an apo protein that has, according to circular dichroism spectroscopy, an alpha-helical content different from that of the holo b type protein. The latter is readily reformed in vitro by addition of heme to the apo protein. This reforming suggests that previously observed assembly of cytochrome c(552), which has the typical class I cytochrome c fold, in the E. coli cytoplasm is a consequence of spontaneous thioether bond formation after binding of heme to a prefolded polypeptide. These observations have implications for the general problem of c type cytochrome biogenesis.     

Resolution of the electronic transitions of cytochrome c oxidase: evidence for two conformational states of ferrous cytochrome alpha.

Second-derivative absorption spectra are reported for a variety of oxidation and ligation states of bovine cytochrome c oxidase (ferrocytochrome-c:oxygen oxidoreductase, EC 1.9.3.1). The high resolving power of the second-derivative method allows us to assign the individual electronic transitions of cytochrome alpha and cytochrome alpha 3 in many of these states. In the fully reduced enzyme, one observes a single electronic transition at 444 nm, corresponding to the Soret transition for both ferrous cytochrome alpha and ferrous cytochrome alpha 3. When the cytochrome alpha 3 site is occupied by an exogenous ligand (CN or CO), one observes two absorption bands assignable to the ferrous cytochrome alpha chromophore, one at ca, 443 nm and the other at ca, 450 nm. The appearance of the 450-nm band is dependent only on ligand occupancy at the cytochrome alpha 3 site and not on the oxidation state of the cytochrome alpha 3 iron. These results can be interpreted either in terms of a heterogeneous mixture of two ferrous cytochrome alpha conformers in the cytochrome alpha 3-ligated enzyme or in terms of a reduction in the effective molecular symmetry of the ferrous cytochrome alpha site that results in a lifting of the degeneracy of the lowest unoccupied molecular orbital associated with the Soret pi,pi* transition of cytochrome alpha. In either case, the present data indicate that ferrous cytochrome alpha can adopt two distinct conformations. One possible structural difference between these two states could be related to differences in the strength of hydrogen bonding between the ferrous cytochrome alpha formyl oxygen and a proton donor from an unidentified amino acid side chain of the enzyme. The implications of such modulation of hydrogen-bond strength are discussed in terms of possible mechanisms of proton translocation and electron transfer in the enzyme.     

Pseudomonas cytochrome c551 at 2.0 A resolution: enlargement of the cytochrome c family.

The structure of respiratory cytochrome c551 of Pseudomonas aeruginosa, with 82 amino acids, has been solved by x-ray analysis and refined to a crystallographic R factor of 16.2%. It has the same basic folding pattern and hydrophobic heme environment as cytochromes c, c2, and c550, except for a large deletion at the bottom of the heme crevice. This same "cytochrome fold" appears to be present in photosynthetic cytochromes c of green and purple sulfur bacteria, and algal cytochromes f, suggesting a common evolutionary origin for electron transport chains in photosynthesis and respiration.     

Kinetic evidence for an on-pathway intermediate in the folding of cytochrome c

An early folding event of cytochrome c populates a helix-containing intermediate (I_NC) because of a pH-dependent misligation between the heme iron and nonnative ligands in the unfolded state (U). For folding to proceed, the nonnative ligation error must first be corrected. It is not known whether I is on-pathway, with folding to the native state (N) as in U ↔ I_NC ↔ N, or whether the I must first move back through the U and then fold to the N through some alternative path (I_NC ↔ U ↔ N). By means of a kinetic test, it is shown here that the cytochrome c I does not first unfold to U. The method used provides an experimental criterion for rejecting the off-pathway I ↔ U ↔ N option.     

The low-spin heme of cytochrome c oxidase as the driving element of the proton-pumping process

Mitochondrial cytochrome c oxidase plays an essential role in aerobic cellular respiration, reducing dioxygen to water in a process coupled with the pumping of protons across the mitochondrial inner membrane. An aspartate residue, Asp-51, located near the enzyme surface, undergoes a redox-coupled x-ray structural change, which is suggestive of a role for this residue in redox-driven proton pumping. However, functional or mechanistic evidence for the involvement of this residue in proton pumping has not yet been obtained. We report that the Asp-51 --> Asn mutation of the bovine enzyme abolishes its proton-pumping function without impairment of the dioxygen reduction activity. Improved x-ray structures (at 1.8/1.9-A resolution in the fully oxidized/reduced states) show that the net positive charge created upon oxidation of the low-spin heme of the enzyme drives the active proton transport from the interior of the mitochondria to Asp-51 across the enzyme via a water channel and a hydrogen-bond network, located in tandem, and that the enzyme reduction induces proton ejection from the aspartate to the mitochondrial exterior. A peptide bond in the hydrogen-bond network critically inhibits reverse proton transfer through the network. A redox-coupled change in the capacity of the water channel, induced by the hydroxyfarnesylethyl group of the low-spin heme, suggests that the channel functions as an effective proton-collecting region. Infrared results indicate that the conformation of Asp-51 is controlled only by the oxidation state of the low-spin heme. These results indicate that the low-spin heme drives the proton-pumping process.     

Water molecule reorganization in cytochrome c oxidase revealed by FTIR spectroscopy

Although internal electron transfer and oxygen reduction chemistry in cytochrome c oxidase are fairly well understood, the associated groups and pathways that couple these processes to gated proton translocation across the membrane remain unclear. Several possible pathways have been identified from crystallographic structural models; these involve hydrophilic residues in combination with structured waters that might reorganize to form transient proton transfer pathways during the catalytic cycle. To date, however, comparisons of atomic structures of different oxidases in different redox or ligation states have not provided a consistent answer as to which pathways are operative or the details of their dynamic changes during catalysis. In order to provide an experimental means to address this issue, FTIR spectroscopy in the 3,560-3,800 cm(-1) range has been used to detect weakly H-bonded water molecules in bovine cytochrome c oxidase that might change during catalysis. Full redox spectra exhibited at least four signals at 3,674(+), 3,638(+), 3,620(-), and 3,607(+) cm(-1). A more complex set of signals was observed in spectra of photolysis of the ferrous-CO compound, a reaction that mimics the catalytic oxygen binding step, and their D(2)O and H(2)(18)O sensitivities confirmed that they arose from water molecule rearrangements. Fitting with Gaussian components indicated the involvement of up to eight waters in the photolysis transition. Similar signals were also observed in photolysis spectra of the ferrous-CO compound of bacterial CcO from Paracoccus denitrificans. Such water changes are discussed in relation to roles in hydrophilic channels and proton/electron coupling mechanism.     

Resonance Raman spectra of hemoglobin and cytochrome c: inverse polarization and vibronic scattering

Resonance Raman spectra of hemoglobin and cytochrome c in dilute solution contain prominent bands that exhibit inverse polarization, i.e., the polarization vector of the incident radiation is rotated through 90 degrees for 90 degrees scattering, giving infinite depolarization ratios. This phenomenon is shown to require an antisymmetric molecular-scattering tensor. The antisymmetry, which is characteristic of resonance scattering, is associated with the form of a particular class of vibrations, A(20), of the tetragonal heme chromophores. The dependence of the resonance Raman spectra on the wavelength of the exciting radiation, as well as their polarization properties, demonstrates that the prominent bands correspond to vibronically active modes of the first electronic transition of the heme proteins, and provide confirmation of Albrecht's vibronic theory of Raman intensities.     

Subpicosecond oxygen trapping in the heme pocket of the oxygen sensor FixL observed by time-resolved resonance Raman spectroscopy

Dissociation of oxygen from the heme domain of the bacterial oxygen sensor protein FixL constitutes the first step in hypoxia-induced signaling. In the present study, the photodissociation of the heme-O2 bond was used to synchronize this event, and time-resolved resonance Raman (TR(3)) spectroscopy with subpicosecond time resolution was implemented to characterize the heme configuration of the primary photoproduct. TR(3) measurements on heme-oxycomplexes are highly challenging and have not yet been reported. Whereas in all other known six-coordinated heme protein complexes with diatomic ligands, including the oxymyoglobin reported here, heme iron out-of-plane motion (doming) occurs faster than 1 ps after iron-ligand bond breaking; surprisingly, no sizeable doming is observed in the oxycomplex of the Bradyrhizobium japonicum FixL sensor domain (FixLH). This assessment is deduced from the absence of the iron-histidine band around 217 cm(-1) as early as 0.5 ps. We suggest that efficient ultrafast oxygen rebinding to the heme occurs on the femtosecond time scale, thus hindering heme doming. Comparing WT oxy-FixLH, mutant proteins FixLH-R220H and FixLH-R220Q, the respective carbonmonoxy-complexes, and oxymyoglobin, we show that a hydrogen bond of the terminal oxygen atom with the residue in position 220 is responsible for the observed behavior; in WT FixL this residue is arginine, crucially implicated in signal transmission. We propose that the rigid O2 configuration imposed by this residue, in combination with the hydrophobic and constrained properties of the distal cavity, keep dissociated oxygen in place. These results uncover the origin of the "oxygen cage" properties of this oxygen sensor protein.     

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.