Origin of Enantioselectivity in Engineered Cytochrome c-Catalyzed Carbon-Radical FePP Hydrolysis Revealed Using QM/MM (ABEEM Polarizable Force Field) and MD Simulations.

The origin of highly efficient asymmetric aminohydroxylation of styrene catalyzed by engineered cytochrome c is investigated by the developed Atom-Bond Electronegativity Equalization Method polarizable force field (ABEEM PFF), which is a combined outcome of electronic and steric effects. Model molecules were used to establish the charge parameters of the ABEEM PFF, for which the bond-stretching and angle-bending parameters were obtained by using a combination of modified Seminario and scan methods. The interactions between carbon-radical Fe-porphyrin (FePP) and waters are simulated by molecular dynamics, which shows a clear preference for the pre-R over the pre-S. This preference is attributed to the hydrogen-bond between the mutated 100S and 101P residues as well as van der Waals interactions, enforcing a specific conformation of the carbon-radical FePP complex within the binding pocket. Meanwhile, the hydrogen-bond between water and the nitrogen atom in the active intermediate dictates the stereochemical outcome. Quantum mechanics/molecular mechanics (QM/MM (ABEEM PFF)) and free-energy perturbation calculations elucidate that the 3RTS is characterized by sandwich-like structure among adjacent amino acid residues, which exhibits greater stability than crowed arrangement in 3STS and enables the R enantiomer to form more favorably. Thus, this study provides mechanistic insight into the catalytic reaction of hemoproteins.

Pulsed Direct Current Arc-Induced Nanoelectrospray Ionization Mass Spectrometry.

This study explores the innovative field of pulsed direct current arc-induced nanoelectrospray ionization mass spectrometry (DCAI-nano-ESI-MS), which utilizes a low-temperature direct current (DC) arc to induce ESI during MS analyses. By employing a 15 kV output voltage, the DCAI-nano-ESI source effectively identifies various biological molecules, including angiotensin II, bradykinin, cytochrome C, and soybean lecithin, showcasing impressive analyte signals and facilitating multicharge MS in positive- and negative-ion modes. Notably, results show that the oxidation of fatty acids using a DC arc produces [M + O - H]- ions, which aid in identifying the location of C═C bonds in unsaturated fatty acids and distinguishing between isomers based on diagnostic ions observed during collision-induced dissociation tandem MS. This study presents an approach for identifying the sn-1 and sn-2 positions in phosphatidylcholine using phosphatidylcholine and nitrate adduct ions, accurately determining phosphatidylcholine molecular configurations via the Paternò-Büchi reaction. With all the advantages above, DCAI-nano-ESI holds significant promise for future analytical and bioanalytical applications.

Deciphering the Roles of Interfacial Amino Acids in Inter-Protein Charge Transport.

Elucidating charge transport (CT) through proteins is critical for gaining insights into ubiquitous CT chain reactions in biological systems and developing high-performance bioelectronic devices. While intra-protein CT has been extensively studied, crucial knowledge about inter-protein CT via interfacial amino acids is still absent due to the structural complexity. Herein, by loading cytochrome c (Cyt c) on well-defined peptide self-assembled monolayers to mimic the protein-protein interface, we provide a precisely controlled platform for identifying the roles of interfacial amino acids in solid-state CT via peptide-Cyt c junctions. The terminal amino acid of peptides serves as a fine-tuning factor for both the interfacial interaction between peptides and Cyt c and the immobilized Cyt c orientation, resulting in a nearly 10-fold difference in current through peptide-Cyt c junctions with varied asymmetry. This work provides a valuable platform for studying CT across proteins and contributes to the understanding of fundamental principles governing inter-protein CT.

Situating the phosphonated calixarene-cytochrome C association by molecular dynamics simulations.

Protein-calixarenes binding plays an increasingly central role in many applications, spanning from molecular recognition to drug delivery strategies and protein inhibition. These ligands obey a specific bio-supramolecular chemistry, which can be revealed by computational approaches, such as molecular dynamics simulations. In this paper, we rely on all-atom, explicit-solvent molecular dynamics simulations to capture the electrostatically driven association of a phosphonated calix-[4]-arene with cytochome-C, which critically relies on surface-exposed paired lysines. Beyond two binding sites identified in direct agreement with the x-ray structure, the association has a larger structural impact on the protein dynamics. Then, our simulations allow a direct comparison to analogous calixarenes, namely, sulfonato, similarly reported as "molecular glue." Our work can contribute to a robust in silico predictive tool to assess binding sites for any given protein of interest for crystallization, with the specificity of a macromolecular cage whose endo/exo orientation plays a role in the binding.

Protective role of chlorogenic acid in preserving cytochrome-c stability against HFIP-induced molten globule state at physiological pH.

Numerous neurodegenerative disorders are characterized by protein misfolding and aggregation. The mechanism of protein aggregation is intricate, and it is very challenging to study at cellular level. Inhibition of protein aggregation by interfering with its pathway is one of the ways to prevent neurodegenerative diseases. In the present work, we have evaluated the protective effect of a polyphenol compound chlorogenic acid (CGA) on the native and molten globule state of horse heart cytochrome c (cyt c). A molten globule state of this heme protein was achieved in the presence of fluorinated alcohol 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) at physiological pH, as studied by UV-Vis absorption, circular dichroism, intrinsic and ANS fluorescence. We found that at 50 % (v/v) HFIP, the native cyt c transformed into a molten globule state. The same techniques were also used to analyze the protective effect of CGA on the molten globule state of cyt c, and the results show that the CGA prevented the molten globular state and retained the protein close to the native state at 1:1 protein:CGA sub molar ratio. Molecular dynamics study also revealed that CGA retains the stability of cyt c in HFIP medium by preserving it in an intermediate state close to native conformation.

The structure of the diheme cytochrome c4 from Neisseria gonorrhoeae reveals multiple contributors to tuning reduction potentials.

Cytochrome c4 (c4) is a diheme protein implicated as an electron donor to cbb3 oxidases in multiple pathogenic bacteria. Despite its prevalence, understanding of how specific structural features of c4 optimize its function is lacking. The human pathogen Neisseria gonorrhoeae (Ng) thrives in low oxygen environments owing to the activity of its cbb3 oxidase. Herein, we report characterization of Ng c4. Spectroelectrochemistry experiments of the wild-type (WT) protein have shown that the two Met/His-ligated hemes differ in potentials by ∼100 mV, and studies of the two His/His-ligated variants provided unambiguous assignment of heme A from the N-terminal domain of the protein as the high-potential heme. The crystal structure of the WT protein at 2.45 Å resolution has revealed that the two hemes differ in their solvent accessibility. In particular, interactions made by residues His57 and Ser59 in Loop1 near the axial ligand Met63 contribute to the tight enclosure of heme A, working together with the surface charge, to raise the reduction potential of the heme iron in this domain. The structure reveals a prominent positively-charged patch, which encompasses surfaces of both domains. In contrast to prior findings with c4 from Pseudomonas stutzeri, the interdomain interface of Ng c4 contributes minimally to the values of the heme iron potentials in the two domains. Analyses of the heme solvent accessibility, interface properties, and surface charges offer insights into the interplay of these structural elements in tuning redox properties of c4 and other multiheme proteins.

Real-Time Observation of Conformational Changes and Translocation of Endogenous Cytochrome c within Intact Mitochondria.

Cytochrome c (cyt c) is a multifunctional protein with varying conformations. However, the conformation of cyt c in its native environment, mitochondria, is still unclear. Here, we applied NMR spectroscopy to investigate the conformation and location of endogenous cyt c within intact mitochondria at natural isotopic abundance, mainly using widespread methyl groups as probes. By monitoring time-dependent chemical shift perturbations, we observed that most cyt c is located in the inner mitochondrial membrane and partially unfolded, which is distinct from its native conformation in solution. When suffering oxidative stress, cyt c underwent oxidative modifications due to increasing reactive oxygen species (ROS), weakening electrostatic interactions with the membrane, and gradually translocating into the inner membrane spaces of mitochondria. Meanwhile, the lethality of oxidatively modified cyt c to cells was reduced compared with normal cyt c. Our findings significantly improve the understanding of the molecular mechanisms underlying the regulation of ROS by cyt c in mitochondria. Moreover, it highlights the potential of NMR to monitor high-concentration molecules at a natural isotopic abundance within intact cells or organelles.

Assessing Lanthanide-Dependent Methanol Dehydrogenase Activity: The Assay Matters.

Artificial dye-coupled assays have been widely adopted as a rapid and convenient method to assess the activity of methanol dehydrogenases (MDH). Lanthanide(Ln)-dependent XoxF-MDHs are able to incorporate different lanthanides (Lns) in their active site. Dye-coupled assays showed that the earlier Lns exhibit a higher enzyme activity than the late Lns. Despite widespread use, there are limitations: oftentimes a pH of 9 and activators are required for the assay. Moreover, Ln-MDH variants are not obtained by isolation from the cells grown with the respective Ln, but by incubation of an apo-MDH with the Ln. Herein, we report the cultivation of Ln-dependent methanotroph Methylacidiphilum fumariolicum SolV with nine different Lns, the isolation of the respective MDHs and the assessment of the enzyme activity using the dye-coupled assay. We compare these results with a protein-coupled assay using its physiological electron acceptor cytochrome cGJ (cyt cGJ ). Depending on the assay, two distinct trends are observed among the Ln series. The specific enzyme activity of La-, Ce- and Pr-MDH, as measured by the protein-coupled assay, exceeds that measured by the dye-coupled assay. This suggests that early Lns also have a positive effect on the interaction between XoxF-MDH and its cyt cGJ thereby increasing functional efficiency.

Effect of proline content and histidine ligation on the dynamics of Ω-loop D and the peroxidase activity of iso-1-cytochrome c.

To study how proline residues affect the dynamics of Ω-loop D (residues 70 to 85) of cytochrome c, we prepared G83P and G83A variants of yeast iso-1-cytochrome c (iso-1-Cytc) in the presence and absence of a K73H mutation. Ω-loop D is important in controlling both the electron transfer function of Cytc and the peroxidase activity of Cytc used in apoptosis because it provides the Met80 heme ligand. The G83P and G83A mutations have no effect on the global stability of iso-1-Cytc in presence or absence of the K73H mutation. However, both mutations destabilize the His73-mediated alkaline conformer relative to the native state. pH jump stopped-flow experiments show that the dynamics of the His73-mediated alkaline transition are significantly enhanced by the G83P mutation. Gated electron transfer studies show that the enhanced dynamics result from an increased rate of return to the native state, whereas the rate of loss of Met80 ligation is unchanged by the G83P mutation. Thus, the G83P substitution does not stiffen the conformation of the native state. Because bis-His heme ligation occurs when Cytc binds to cardiolipin-containing membranes, we studied the effect of His73 ligation on the peroxidase activity of Cytc, which acts as an early signal in apoptosis by causing oxygenation of cardiolipin. We find that the His73 alkaline conformer suppresses the peroxidase activity of Cytc. Thus, the bis-His ligated state of Cytc formed upon binding to cardiolipin is a negative effector for the peroxidase activity of Cytc early in apoptosis.

Effects of removal of the axial methionine heme ligand on the binding of S. cerevisiae iso-1 cytochrome c to cardiolipin.

The cleavage of the axial S(Met) - Fe bond in cytochrome c (cytc) upon binding to cardiolipin (CL), a glycerophospholipid of the inner mitochondrial membrane, is one of the key molecular changes that impart cytc with (lipo)peroxidase activity essential to its pro-apoptotic function. In this work, UV - VIS, CD, MCD and fluorescence spectroscopies were used to address the role of the Fe - M80 bond in controlling the cytc-CL interaction, by studying the binding of the Met80Ala (M80A) variant of S. cerevisiae iso-1 cytc (ycc) to CL liposomes in comparison with the wt protein [Paradisi et al. J. Biol. Inorg. Chem. 25 (2020) 467-487]. The results show that the integrity of the six-coordinate heme center along with the distal heme site containing the Met80 ligand is a not requisite for cytc binding to CL. Indeed, deletion of the Fe - S(Met80) bond has a little impact on the mechanism of ycc-CL interaction, although it results in an increased heme accessibility to solvent and a reduced structural stability of the protein. In particular, M80A features a slightly tighter binding to CL at low CL/cytc ratios compared to wt ycc, possibly due to the lift of some constraints to the insertion of the CL acyl chains into the protein hydrophobic core. M80A binding to CL maintains the dependence on the CL-to-cytc mixing scheme displayed by the wt species.

Suitability of Adenosine Derivatives in Improving the Activity and Stability of Cytochrome c under Stress: Insights into the Effect of Phosphate Groups.

It is well known that adenosine and its phosphate derivatives play a crucial role in biological phenomena such as apoptosis and cell signaling and act as the energy currency of the cell. Although their interactions with various proteins and enzymes have been described, the focus of this work is to demonstrate the effect of the phosphate group on the activity and stability of the native heme metalloprotein cytochrome c (Cyt c), which is important from both biological and industrial aspects. In situ and in silico characterizations are used to correlate the relationship between the binding affinity of adenosine and its phosphate groups with unfolding behavior, corresponding peroxidase activities, and stability factors. Interaction of adenosine (ADN), adenosine monophosphate (AMP), adenosine 5'-diphosphate (ADP), and adenosine 5'-triphosphate (ATP) with Cyt c increases peroxidase-like activity by up to 1.8-6.5-fold compared to native Cyt c. This activity is significantly maintained even after multiple stress conditions such as oxidative stress and the presence of a chaotropic agent such as guanidine hydrochloride (GuHCl). With binding affinities on the order of ADN < AMP < ADP < ATP, adenosine derivatives were found to stabilize Cyt c by varying the secondary structural features of the protein. Thus, in addition to being a fundamental study, the current work also proposes a way of stabilizing protein systems to be used for real-time biocatalytic applications.

In Situ Raman Spectroscopy Reveals Cytochrome c Redox-Controlled Modulation of Mitochondrial Membrane Permeabilization That Triggers Apoptosis.

The selective interaction of cytochrome c (Cyt c) with cardiolipin (CL) is involved in mitochondrial membrane permeabilization, an essential step for the release of apoptosis activators. The structural basis and modulatory mechanism are, however, poorly understood. Here, we report that Cyt c can induce CL peroxidation independent of reactive oxygen species, which is controlled by its redox states. The structural basis of the Cyt c-CL binding was unveiled by comprehensive spectroscopic investigation and mass spectrometry. The Cyt c-induced permeabilization and its effect on membrane collapse, pore formation, and budding are observed by confocal microscopy. Moreover, cytochrome c oxidase dysfunction is found to be associated with the initiation of Cyt c redox-controlled membrane permeabilization. These results verify the significance of a redox-dependent modulation mechanism at the early stage of apoptosis, which can be exploited for the design of cytochrome c oxidase-targeted apoptotic inducers in cancer therapy.

Contribution of a surface salt bridge to the protein stability of deep-sea Shewanella benthica cytochrome c'.

Two homologous cytochromes c', SBCP and SVCP, from deep-sea Shewanella benthica and Shewanella violacea respectively exhibit only nine surface amino acid substitutions, along with one at the N-terminus. Despite the small sequence difference, SBCP is thermally more stable than SVCP. Here, we examined the thermal stability of SBCP variants, each containing one of the nine substituted residues in SVCP, and found that the SBCP K87V variant was the most destabilized. We then determined the X-ray crystal structure of the SBCP K87V variant at a resolution of 2.1 Å. The variant retains a four-helix bundle structure similar to the wild-type, but notable differences are observed in the hydration structure around the mutation site. Instead of forming of the intrahelical salt bridge between Lys-87 and Asp-91 in the wild-type, a clathrate-like hydration around Val-87 through a hydrogen bond network with the nearby amino acid residues is observed. This network potentially enhances the ordering of surrounding water molecules, leading to an entropic destabilization of the protein. These results suggest that the unfavorable hydrophobic hydration environment around Val-87 and the inability to form the Asp-91-mediated salt bridge contribute to the observed difference in stability between SBCP and SVCP. These findings will be useful in future protein engineering for controlling protein stability through the manipulation of surface intrahelical salt bridges.

Ferrous nitrosylated cytochrome c: The unusual strength of the proximal His18-Fe bond.

NO binding to horse heart cytochrome c (hhcyt c) has been investigated as a function of pH by both optical absorption and EPR spectroscopies. Lowering pH from 3.5 to 1.5 induces: (i) a blue-shift of the maximum of the optical absorption spectrum in the Soret region from 415 to about 404 nm, and (ii) the appearance of a strong three hyperfine splitting in the gz region of the EPR spectrum. Both spectroscopic features indicate the cleavage of the proximal His18-Fe(II)-NO bond giving rise to the five-coordinated Fe(II)-NO species. By quantification of the relative weight for the six- and the five-coordinated component in the EPR spectra, the pKa value was determined. The apparent pKa of the proximal His Nε atom (1.8 ±â€¯0.1) is unusually low for a ferrous nitrosylated form since in all investigated ferrous NO-bound heme-proteins the pKa value for the cleavage of the proximal His-Fe(II) bond ranges between 3.7 and 5.8. The pKa value of ferrous nitrosylated hhcyt c indicates that the strength of the proximal His18-Fe(II) bond (= 27.9 kJ/mol) is about 10-22 kJ/mol higher than that observed in all investigated heme-proteins. The strong coordination of the heme-Fe atom by His18 is extremely important to maintain the redox efficiency of cyt c and to keep apoptosis under control. This is a crucial point in tissues, such as retina, where apoptosis might trigger macular degenerative processes.

A π-π Bonding-Assisted Molecular-Wiring of Folded-Cytochrome c and Naphthoquinone and Its Electron-Relay-Based Bioelectrocatalytic H2O2 Reduction Reaction Visualized by Redox-Competitive Scanning Electrochemical Microscopy.

The electron-transfer (ET) reaction of cytochrome c (Cytc) protein with biomolecules is a cutting-edge research area of interest in understanding the functionalities of natural systems. Several electrochemical biomimicking studies based on Cytc-protein-modified electrodes prepared via electrostatic interaction and covalent bonding approaches have been reported. Indeed, natural enzymes involve multiple types of bonding, such as hydrogen, ionic, covalent, and π-π, etc. In this work, we explore a Cytc-protein chemically modified glassy carbon electrode (GCE/CB@NQ/Cytc) prepared via π-π bonding using graphitic carbon as an underlying surface and an aromatic organic molecule, naphthoquinone (NQ), as a cofactor for an effective ET reaction. A simple drop-casting technique-based preparation of GCE/CB@NQ showed a distinct surface-confined redox peak at a standard electrode potential (E°) = -0.2 V vs Ag/AgCl (surface excess = 21.3 nmol cm-2) in pH 7 phosphate buffer solution. A control experiment of modification of NQ on an unmodified GCE failed to show any such unique feature. For the preparation of GCE/CB@NQ/Cytc, a dilute solution of Cytc-pH 7 phosphate buffer was drop-cast on the GCE/CB@NQ surface, wherein the protein folding and denaturalization-based complication and its associated ET functionalities were avoided. Molecular dynamics simulation studies show the complexation of NQ with Cytc at the protein binding sites. The protein-bound surface shows an efficient and selective bioelectrocatalytic reduction performance of H2O2, as demonstrated using cyclic voltammetry and amperometric i-t techniques. Finally, the redox-competition scanning electrochemical microscopy (RC-SECM) technique was adopted for in situ visualization of the electroactive adsorbed surface. The RC-SECM images clearly show the regions of highly bioelectrocatalytic active sites of Cytc-proteins bound to NQ molecules on a graphitic carbon surface. The binding of Cytc with NQ has significant implications for studying the biological electron transport mechanism, and the proposed method provides the requisite framework for such a study.

Effects of naturally occurring S47F/A mutations on the structure and function of human cytochrome c.

The sequence and structure of human cytochrome c (hCyt c) exhibit evolutionary conservations, with only a limited number of naturally occurring mutations in humans. Herein, we investigated the effects of the naturally occurring S47F/A mutations on the structure and function of hCyt c in the oxidized form. Although the naturally occurring S47F/A mutations did not largely alter the protein structure, the S47F and S47A variants exhibited a small fraction of high-spin species. Kinetic studies showed that the peroxidase activity of the variants was enhanced by ∼2.5-fold under neutral pH conditions, as well as for the rate in reaction with H2O2, when compared to those of wild-type hCyt c. In addition, we evaluated the interaction between hCyt c and human neuroglobin (hNgb) by isothermal titration calorimetry (ITC) studies, which revealed that the binding constant was reduced by ∼8-fold as result of the mutation of the hydrophilic Ser to the hydrophobic Phe/Ala. These findings provide valuable insights into the role of Ser47 in Ω-loop C in sustaining the structure and function of hCyt c.

Are the Gas-Phase Structures of Molecular Elephants Enduring or Ephemeral? Results from Time-Dependent, Tandem Ion Mobility.

The structural stability of biomolecules in the gas phase remains an important topic in mass spectrometry applications for structural biology. Here, we evaluate the kinetic stability of native-like protein ions using time-dependent, tandem ion mobility (IM). In these tandem IM experiments, ions of interest are mobility-selected after a first dimension of IM and trapped for up to ∼14 s. Time-dependent, collision cross section distributions are then determined from separations in a second dimension of IM. In these experiments, monomeric protein ions exhibited structural changes specific to both protein and charge state, whereas large protein complexes did not undergo resolvable structural changes on the timescales of these experiments. We also performed energy-dependent experiments, i.e., collision-induced unfolding, as a comparison for time-dependent experiments to understand the extent of unfolding. Collision cross section values observed in energy-dependent experiments using high collision energies were significantly larger than those observed in time-dependent experiments, indicating that the structures observed in time-dependent experiments remain kinetically trapped and retain some memory of their solution-phase structure. Although structural evolution should be considered for highly charged, monomeric protein ions, these experiments demonstrate that higher-mass protein ions can have remarkable kinetic stability in the gas phase.

New Insights into the Intrinsic Electron-Based Dissociation Behavior of Cytochrome c Oligomers.

Between 2003 and 2017, four reports were published that demonstrated the intrinsic ability of the native iron-containing proteins cytochrome c and ferritin to undergo radical-based backbone fragmentation in the gas phase without the introduction of exogenous electrons. For cytochrome c in particular, this effect has so far only been reported to occur in the ion source, preventing the in-depth study of reactions occurring after gas-phase isolation of specific precursors. Here, we report the first observation of this intrinsic native electron capture dissociation behavior after quadrupole isolation of specific charge states of the cytochrome c dimer and trimer, providing direct experimental support for key aspects of the mechanism proposed 20 years ago. Furthermore, we provide evidence that, in contrast to some earlier proposals, these oligomeric states are formed in bulk solution rather than during the electrospray ionization process and that the observed fragmentation site preferences can be rationalized through the structure and interactions within these native oligomers rather than the monomer. We also show that the observed fragmentation pattern─and indeed, whether or not fragmentation occurs─is highly sensitive to the provenance and history of the protein samples, to the extent that samples can show distinct fragmentation behavior despite behaving identically in ion mobility experiments. This rather underexplored method therefore represents an exquisitely sensitive conformational probe and will hopefully receive more attention from the biomolecular mass spectrometry community in the future.

Pulse Duration Effects on Solution-Phase Protein Desorption in Laser Electrospray Mass Spectrometry.

The effect of laser pulse duration on the ablation of aqueous myoglobin is investigated using laser electrospray mass spectrometry (LEMS). Pulse durations of 55 femtoseconds (fs), 56 piscoseconds (ps), and 10 nanoseconds (ns) were used to ablate aqueous myoglobin from stainless-steel and quartz substrates. The integrated signal intensity of myoglobin increases with decreasing pulse duration for both substrates. Laser-induced thermal effects are assessed by the relative amount of solvent adduction and number of phosphate moieties adducted to myoglobin by each laser pulse duration. The mass spectra for 55 fs vaporization shows myoglobin with appreciable solvent and phosphate adduction and baseline elevation. The mass spectra for 10 ns ablation have minimal adduction and limited baseline elevation. Heat-induced conformation changes in myoglobin were used to measure the amount of thermal energy deposited by each laser pulse duration. Ablation using the 55 fs pulse revealed the highest ratio of unfolded to folded myoglobin in comparison to the 56 ps and 10 ns measurements due to increased droplet lifetime and consequent interaction with the acid in the electrospray solvent. Collisional activation and heated capillary temperature were employed to reduce the droplet lifetime and demonstrate that fs ablation preserves approximately 2 times more myoglobin folded conformation in comparison to ps and ns pulses.

Resonance Raman Studies on Heme Ligand Stretching Modes in Methionine80-Depleted Cytochrome c: Fe-His, Fe-O2, and O-O Stretching Modes.

The peroxidase activity of cytochrome (cyt) c increases when Met80 dissociates from the heme iron, which is related to the initial cyt c membrane permeation step of apoptosis. Met80-dissociated cyt c can form an oxygenated species. Herein, resonance Raman spectra of Met80-depleted horse cyt c (M80A cyt c) were analyzed to elucidate the heme ligand properties of Met80-dissociated cyt c. The Fe-His stretching (νFe-His) mode of ferrous M80A cyt c was observed at 236 cm-1, and this frequency decreased by 1.5 cm-1 for the 15N-labeled protein. The higher νFe-His frequency of M80A cyt c than of other His-ligated heme proteins indicates strong heme coordination and the imidazolate character of His18. Peaks attributed to the Fe-O2 stretching (νFe-O2) and O-O stretching (νO-O) modes of the oxygenated species of M80A cyt c were observed at 576 and 1148 cm-1, respectively, under an 16O2 atmosphere, whereas the frequencies decreased to 544 and 1077 cm-1, respectively, under an 18O2 atmosphere. The νFe-O2 mode of Hydrogenobacter thermophilus (HT) M59A cyt c552 was observed at 580 cm-1 under an 16O2 atmosphere, whereas the frequency decreased to 553 cm-1 under an 18O2 atmosphere, indicating that relatively high νFe-O2 frequencies are characteristic of c-type cyt proteins. By comparison of the simultaneously observed νFe-O2 and νO-O frequencies of oxygenated cyt c and other oxygenated His-ligated heme proteins, the frequencies tend to have a positive linear relationship; the νFe-O2 frequency increases when the νO-O frequency increases. The imidazolate character of the heme-coordinated His and strong Fe-O and O-O bonds are characteristic of cyt c and apparently related to the peroxidase activity when Met80 dissociates from the heme iron.