Methionine Ligand Research Articles

Substitution of the Axial Type 1 Cu Ligand Affords Binding of a Water Molecule in Axial Position Affecting Kinetics, Spectral, and Structural Properties of the Small Laccase Ssl1.

Multicopper oxidases use Cu ions as cofactors to oxidize various substrates. High reduction potential at Type 1 Cu is considered as crucial for effective catalysis. Previous studies have shown that replacing the axial methionine ligand of the Type 1 Cu with leucine or phenylalanine leads to an increased reduction potential, but not always to higher enzyme activity. Here we present a study on six variants of the small laccase Ssl1 from Streptomyces sviceus, where the axial methionine ligand was substituted, and the effect of the axial ligand on reduction potential, activity, spectral properties and structure was investigated. Absorption, electronic circular dichroism and EPR spectra revealed the presence of a stronger coordinating axial ligand like oxygen, which influences the electronic and catalytic properties more than the nature of the amino acid side chain. The crystal structures of the Ssl1 variants were solved, which show that none of the amino acid side chains coordinate to the Cu. Instead, a water molecule is found in the axial coordination site, which support the spectroscopic data. Our findings highlight the importance of combining structural and spectroscopic methods to investigate the effect of amino acid exchange on multicopper oxidases.

In silico Characterization of a Hypothetical Protein (PBJ89160.1) from Neisseria meningitidis Exhibits a New Insight on Nutritional Virulence and Molecular Docking to Uncover a Therapeutic Target.

Neisseria meningitidis is an encapsulated, diplococcus, kidney bean-shaped bacteria that causes bacterial meningitis. Our study hopes to advance our understanding of disease progression, the spread frequency of the bacteria in people, and the interactions between the bacteria and human body by identifying a functional protein, potentially serving as a target for meningococcal medicine in the future. A hypothetical protein HP (PBJ89160.1) from N. meningitidis was employed in this study for extensive structural and functional characterization. In the predictive functional role of HP, several constitutive bioinformatics approaches are applied, such as prediction of physiological properties, domain and motif family function, secondary and tertiary structure prediction, energy minimization, quality validation, docking, and ADMET analysis. To create the protein's three-dimensional (3D) structure, a template protein (PDB_ID: 3GXA) is used with 99% sequence identity by homology modeling technique with the HHpred server. To mitigate the pathogenicity associated with the HP function, it was docked with the natural ligand methionine and five other drug compounds like Verapamil, Loperamide, Thioridazine, Chlorpromazine, and Auranofine. The protein is predicted to be acidic, soluble and hydrophilic by physicochemical properties analysis. Subcellular localization analysis demonstrated the protein to be periplasmic. The HP has an ATP-binding cassette transporter (also known as ABC transporter) involved in uptake of methionine (MetQ) that creates nutritional virulence in host. Energy minimization, multiple quality assessments, and validation value determination led to the conclusion that the HP model had a workable and acceptable quality. Following ADMET analysis and binding affinity assessments from the docking studies, Loperamide emerged as the most promising therapeutic compound, effectively inhibiting the ATP transporter activity of the HP. Comparative genomic analysis revealed that this protein is specific to N. meningitidis and has no homologs in human proteins, thereby identifying it as a potential target for therapeutic intervention.

The structure of plastocyanin tunes the midpoint potential by restricting axial ligation of the reduced copper ion

Blue copper proteins are models for illustrating how proteins tune metal properties. Nevertheless, the mechanisms by which the protein controls the metal site remain to be fully elucidated. A hindrance is that the closed shell Cu(I) site is inaccessible to most spectroscopic analyses. Carbon deuterium (C-D) bonds used as vibrational probes afford nonperturbative, selective characterization of the key cysteine and methionine copper ligands in both redox states. The structural integrity of Nostoc plastocyanin was perturbed by disrupting potential hydrogen bonds between loops of the cupredoxin fold via mutagenesis (S9A, N33A, N34A), variably raising the midpoint potential. The C-D vibrations show little change to suggest substantial alteration to the Cu(II) coordination in the oxidized state or in the Cu(I) interaction with the cysteine ligand. They rather indicate, along with visible and NMR spectroscopy, that the methionine ligand distinctly interacts more strongly with the Cu(I) ion, in line with the increases in midpoint potential. Here we show that the protein structure determines the redox properties by restricting the interaction between the methionine ligand and Cu(I) in the reduced state.

Contributions to cytochrome c inner- and outer-sphere reorganization energy.

Cytochromes c are small water-soluble proteins that catalyze electron transfer in metabolism and energy conversion processes. Hydrogenobacter thermophilus cytochrome c552 presents a curious case in displaying fluxionality of its heme axial methionine ligand; this behavior is altered by single point mutation of the Q64 residue to N64 or V64, which fixes the ligand in a single configuration. The reorganization energy (λ) of these cytochrome c552 variants is experimentally determined using a combination of rotating disc electrochemistry, chronoamperometry and cyclic voltammetry. The differences between the λ determined from these complementary techniques helps to deconvolute the contribution of the active site and its immediate environment to the overall λ (λTotal). The experimentally determined λ values in conjunction with DFT calculations indicate that the differences in λ among the protein variants are mainly due to the differences in contributions from the protein environment and not just inner-sphere λ. DFT calculations indicate that the position of residue 64, responsible for the orientation of the axial methionine, determines the geometric relaxation of the redox active molecular orbital (RAMO). The orientation of the RAMO with respect to the heme is key to determining electron transfer coupling (HAB) which results in higher ET rates in the wild-type protein relative to the Q64V mutant despite a 150 mV higher λTotal in the former.

First-principles study on the structure and optical spectroscopy of the redox-active center of blue copper proteins

We present first-principles calculations on the geometrical structures and optical absorption properties of redox-active centers in two types of blue copper proteins, plastocyanin (Pc) and pseudoazurin (PAz). Two four-coordination (4C) cluster models consisting 111-atom were constructed based on the crystal structures of the two proteins. The optimized configurations of the proposed models took on highly identical geometry as the crystal structures and the calculated absorption spectra of both models acceptably reproduced the corresponding experimental spectra. Based on spectral simulations, we were able to confirm the indispensable role of axial ligand methionine (Met) in both proteins. The difference in the absorption spectroscopy of the two types of proteins was caused by the changes to the ground state wave functions which originate mainly from the different coordination structures of the redox-active center, especially the different Cu-SMet bond lengths. The effects of density functionals and the size of basis sets were also tested.

Unexpected electron spin density on the axial methionine ligand in CuA suggests its involvement in electron pathways.

The CuA center is a paradigm for the study of long-range biological electron transfer. This metal center is an essential cofactor for terminal oxidases like cytochrome c oxidase, the enzymatic complex responsible for cellular respiration in eukaryotes and in most bacteria. CuA acts as an electron hub by transferring electrons from reduced cytochrome c to the catalytic site of the enzyme where dioxygen reduction takes place. Different electron transfer pathways have been proposed involving a weak axial methionine ligand residue, conserved in all CuA sites. This hypothesis has been challenged by theoretical calculations indicating the lack of electron spin density in this ligand. Here we report an NMR study with selectively labeled methionine in a native CuA. NMR spectroscopy discloses the presence of net electron spin density in the methionine axial ligand in the two alternative ground states of this metal center. Similar spin delocalization observed on two second sphere mutants further supports this evidence. These data provide a novel view of the electronic structure of CuA centers and support previously neglected electron transfer pathways.

Methionine Binding to Dirhodium(II) Tetraacetate.

The reaction between antitumor active dirhodium(II) tetraacetate and dl-methionine (HMet) was followed in aqueous solution and showed initially mixtures of 1:1 and 1:2 adducts [Rh2(AcO)4(HMet)(H2O)] (AcO- = CH3COO-) and [Rh2(AcO)4(HMet)2] formed at room temperature (RT), as evidenced by UV-vis spectroscopy and electrospray ionization mass spectrometry (ESI-MS). Rh K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy confirmed methionine thioether binding to the axial positions of the Rh2(AcO)4 cage structure. With excess HMet at RT, stepwise displacement of the acetate groups was observed after some time using ESI-MS. Heating the solution to 40° for 24 h accelerated the substitution reaction leading to stable dirhodium(II) species with two acetate ligands displaced by two methionine groups. The crystal structure of the purple [RhII2(AcO)2(d-Met)(l-Met)]·6H2O compound obtained from the solution revealed tridentate coordination of the methionine ligands to the Rh(II) ions, with the thioether S atoms in equatorial positions. A minor amount of a light orange monomeric [RhIII(Met)2](AcO) complex also formed in the solution was isolated by size exclusion chromatography and identified by ESI-MS. Crystals of [RhIII(d-Met)(l-Met)]Cl·3H2O were prepared by reacting RhCl3 and dl-HMet. The crystal structure showed tridentate binding of the methionine ligands to the Rh(III) ion in a trans-S, N, O arrangement.

Methionine Ligand Interaction in a Blue Copper Protein Characterized by Site-Selective Infrared Spectroscopy.

The reactivity of metal sites in proteins is tuned by protein-based ligands. For example, in blue copper proteins such as plastocyanin (Pc), the structure imparts a highly elongated bond between the Cu and a methionine (Met) axial ligand to modulate its redox properties. Despite extensive study, a complete understanding of the contribution of the protein to redox activity is challenged by experimentally accessing both redox states of metalloproteins. Using infrared (IR) spectroscopy in combination with site-selective labeling with carbon-deuterium (C-D) vibrational probes, we characterized the localized changes at the Cu ligand Met97 in the oxidized and reduced states, as well as the Zn(II) or Co(II)-substituted, the pH-induced low-coordinate, the apoprotein, and the unfolded states. The IR absorptions of (d3-methyl)Met97 are highly sensitive to interaction of the sulfur-based orbitals with the metal center and are demonstrated to be useful reporters of its modulation in the different states. Unrestricted Kohn-Sham density functional theory calculations performed on a model of the Cu site of Pc confirm the observed dependence. IR spectroscopy was then applied to characterize the impact of binding to the physiological redox partner cytochrome (cyt) f. The spectral changes suggest a slightly stronger Cu-S(Met97) interaction in the complex with cyt f that has potential to modulate the electron transfer properties. Besides providing direct, molecular-level comparison of the oxidized and reduced states of Pc from the perspective of the axial Met ligand and evidence for perturbation of the Cu site properties by redox partner binding, this study demonstrates the localized spatial information afforded by IR spectroscopy of selectively incorporated C-D probes.

Mutation of heme c axial ligands in d-fructose dehydrogenase for investigation of electron transfer pathways and reduction of overpotential in direct electron transfer-type bioelectrocatalysis

d-Fructose dehydrogenase (FDH), a flavoprotein-cytochrome c complex, exhibits high activity in direct electron transfer (DET)-type bioelectrocatalysis. One of the three types of heme c in FDH is the electron-donating site to the electrodes, and another heme c is presumed to not be involved in the catalytic cycle. In order to confirm the electron transfer pathway, we constructed three mutants in which the sixth axial methionine ligand (M301, M450, or M578) of one of the hemes was replaced with glutamine, which was selected with the expectation that it would shift the formal potential of the hemes in the negative direction. An M450Q mutant successfully reduced the overpotential by approximately 0.2V, giving a limiting current close to that of the native FDH. In contrast, an M301Q mutant remained almost unchanged and an M578Q mutant drastically decreased DET-type catalytic activity. The results indicate that the electron transfer in the native FDH occurs in sequence from the flavin, through the heme c with M578, to the heme c with M450 (as the electron-donating site to the electrodes), without going through the heme c with M301. The M450Q mutant will be useful for biofuel cells because of the decreased overpotential.

A Translational Biomedical Approach to Structural Arrangement of Amino Acids Complexes: A Combined Theoretical and Computational Study

In this short communication, respectively bonding among Boron (B3+), Aluminum (Al3+), Gallium (Ga3+), Indium (In3+) and Thallium (Tl3+) cations and Alanine, Arginine, Asparagine, Aspartic Acid, Cysteine, Glutamic Acid, Glutamine, Glycine, Proline, Selenocysteine, Serine, Tyrosine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan and Valine ligands and also structural arrangement of the present compounds were studied, theoretically and computationally

Sequence proximity between Cu(II) and Cu(I) binding sites of human copper transporter 1 model peptides defines reactivity with ascorbate and O2

The critical nature of the copper transporter 1 (Ctr1) in human health has spurred investigation of Ctr1 structure and function. Ctr1 specifically transports Cu(I), the reduced form of copper, across the plasma membrane. Thus, extracellular Cu(II) must be reduced prior to transport. Unlike yeast Ctr1, mammalian Ctr1 does not rely on any known mammalian reductase. Previous spectroscopic studies of model peptides indicate that human Ctr1 could serve as both copper reductase and transporter. Ctr1 peptides bind Cu(II) at an amino terminal high-affinity Cu(II), Ni(II) ATCUN site. Ascorbate-dependent reduction of the Cu(II)–ATCUN complex is possible by virtue of an adjacent HH (bis–His), as this bis–His motif and one methionine ligand constitute a high affinity Ctr1 Cu(I) binding site. Here, we synthetically varied the distance between the ATCUN and bis–His motifs in a series of peptides based on the human Ctr1 amino terminal, with the general sequence MDHAnHHMGMSYMDS, where n=0–4. We tested the ability of each peptide to reduce Cu(II) with ascorbate and stabilize Cu(I) under ambient conditions (20% O2). This study reveals that significant differences in coordination structure and chemical behavior with ascorbate and O2 result from changes in the sequence proximity of ATCUN and bis–His. Peptides that deviate from the native Ctr1 pattern were less effective at forming stable Cu(I)–peptide complexes and/or resulted in O2-dependent oxidative damage to the peptide.

Heme-bound SiaA from Streptococcus pyogenes: Effects of mutations and oxidation state on protein stability

The protein SiaA (HtsA) is part of a heme uptake pathway in Streptococcus pyogenes. In this report, we present the heme binding of the alanine mutants of the axial histidine (H229A) and methionine (M79A) ligands, as well as a lysine (K61A) and cysteine (C58A) located near the heme propionates (based on homology modeling) and a control mutant (C47A). pH titrations gave pKa values ranging from 9.0 to 9.5, close to the value of 9.7 for WT SiaA. Resonance Raman spectra of the mutants suggested that the ferric heme environment may be distinct from the wild-type; spectra of the ferrous states were similar. The midpoint reduction potential of the K61A mutant was determined by spectroelectrochemical titration to be 61±3mV vs. SHE, similar to the wild-type protein (68±3mV). The addition of guanidine hydrochloride showed two processes for protein denaturation, consistent with heme loss from protein forms differing by the orientation of the heme in the binding pocket (the half-life for the slower process ranged from less than half a day to two days). The ease of protein unfolding was related to the strength of interaction of the residues with the heme. We hypothesize that kinetically facile but only partial unfolding, followed by a very slow approach to the completely unfolded state, may be a fundamental attribute of heme trafficking proteins. Small motions to release/transfer the heme accompanied by resistance to extensive unfolding may preserve the three dimensional form of the protein for further uptake and release.

Structure analysis and characterization of the cytochrome c-554 from thermophilic green sulfur photosynthetic bacterium Chlorobaculum tepidum

The cytochrome (Cyt) c-554 in thermophilic green photosynthetic bacterium Chlorobaculum tepidum serves as an intermediate electron carrier, transferring electrons to the membrane-bound Cyt c z from various enzymes involved in the oxidations of sulfide, thiosulfate, and sulfite compounds. Spectroscopically, this protein exhibits an asymmetric α-absorption band for the reduced form and particularly large paramagnetic (1)H NMR shifts for the heme methyl groups with an unusual shift pattern in the oxidized form. The crystal structure of the Cyt c-554 has been determined at high resolution. The overall fold consists of four α-helices and is characterized by a remarkably long and flexible loop between the α3 and α4 helices. The axial ligand methionine has S-chirality at the sulfur atom with its C(ε)H3 group pointing toward the heme pyrrole ring I. This configuration corresponds to an orientation of the lone-pair orbital of the sulfur atom directed at the pyrrole ring II and explains the lowest-field (1)H NMR shift arising from the 18(1) heme methyl protons. Differing from most other class I Cyts c, no hydrogen bond was formed between the methionine sulfur atom and polypeptide chain. Lack of this hydrogen bond may account for the observed large paramagnetic (1)H NMR shifts of the heme methyl protons. The surface-exposed heme pyrrole ring II edge is in a relatively hydrophobic environment surrounded by several electronically neutral residues. This portion is considered as an electron transfer gateway. The structure of the Cyt c-554 is compared with those of other Cyts c, and possible interactions of this protein with its electron transport partners are discussed.

Investigations of the Low-Frequency Spectral Density of Cytochrome c upon Equilibrium Unfolding

The equilibrium unfolding process of ferric horse heart cytochrome c (cyt c), induced by guanidinium hydrochloride (GdHCl), was studied using UV-vis absorption spectroscopy, resonance Raman spectroscopy, and vibrational coherence spectroscopy (VCS). The unfolding process was successfully fit using a three-state model which included the fully folded (N) and unfolded (U) states, along with an intermediate (I) assigned to a Lys bound heme. The VCS spectra revealed for the first time several low-frequency heme modes that are sensitive to cyt c unfolding: γ(a) (~50 cm(-1)), γ(b) (~80 cm(-1)), γ(c) (~100 cm(-1)), and ν(s)(His-Fe-His) at 205 cm(-1). These out-of-plane modes have potential functional relevance and are activated by protein-induced heme distortions. The free energies for the N-I and the I-U transitions at pH 7.0 and 20 °C were found to be 4.6 kcal/M and 11.6 kcal/M, respectively. Imidazole was also introduced to replace the methionine ligand so the unfolding can be modeled as a two-state system. The intensity of the mode γ(b)~80 cm(-1) remains nearly constant during the unfolding process, while the amplitudes of the other low frequency modes track with spectral changes observed at higher frequency. This confirms that the heme deformation changes are coupled to the protein tertiary structural changes that take place upon unfolding. These studies also reveal that damping of the coherent oscillations depends sensitively on the coupling between heme and the surrounding water solvent.

“Strange Kinetics” in the Temperature Dependence of Methionine Ligand Rebinding Dynamics in Cytochrome c

The temperature dependence of methionine ligand dissociation and rebinding dynamics in cytochrome c in aqueous solution has been studied using classical molecular dynamics simulation. Results are compared with previous study of rebinding dynamics at 300 K in water in order to understand how the change of protein environment and the underlying protein energy landscape influence the dynamics. Rebinding dynamics at 77, 180, and 300 K exhibits changes in both time scale and mechanism as the protein and solvent undergo a dynamic "glass transition". At each temperature, the rebinding dynamics yields a subset of trajectories that undergo fast rebinding as well as a subset of trajectories that undergo slower rebinding. At 300 K in water, both a fast (4.0 ps) and slow (14.6 ps) rebinding is observed. While fast rebinding occurs from a "downward" (heme pointing) substate of the methionine, the slow rebinding involves interconversion between an "upward" substate, from which rebinding cannot occur, and the downward substate. At lower temperatures (77 and 180 K), the upward dissociated substate was not observed due to the high barrier imposed by the "frozen" protein structure. However, a slow rebinding phase is observed at both 77 and 180 K and is associated with a process of trapping in downward but "binding forbidden" substates with subsequent slow dynamical conversion to "binding competent" substates from which rebinding is relatively rapid. Distinctive rebinding dynamics at 77 and 180 K suggest that different rebinding time scales are predetermined by the protein and solvent structural arrangement prior to photodissociation, which causes either fast rebinding (about 2 ps) or slow (>50 ps) rebinding. Suggestions for future experiments to further probe the role of dynamic heterogeneity in the kinetics of methionine ligand binding in cytochrome c protein are proposed.

Axial iron coordination and spin state change in a heme c upon electrostatic protein–SAM interaction

A bacterial di-heme cytochrome c binds electrostatically to a gold electrode surface coated with a negatively charged COOH-terminated SAM adopting a sort of 'perpendicular' orientation. Cyclic voltammetry, Resonance Raman and SERRS spectroscopies indicate that the high-potential C-terminal heme center proximal to the SAM's surface undergoes an adsorption-induced swapping of one axial His ligand with a water molecule, which is probably lost in the reduced form, and a low- to high-spin transition. This coordination change for a bis-His ligated heme center upon an electrostatically-driven molecular recognition is as yet unprecedented, as well as the resulting increase in reduction potential. We discuss it in comparison with the known methionine ligand lability in monoheme cytochromes c occurring upon interaction with charged molecular patches. One possible implication of this finding in biological ET is that mobile redox partners do not behave as rigid and invariant bodies, but in the ET complex are subjected to molecular changes and structural fluctuations that affect in a complex way the thermodynamics and the kinetics of the process.

Mixed Ligand Complexes of Gold (III) with Some Amino Acids and Dithiocarbamates or Dithiophosphates

Complexes of the type [Au(L)(L-orL=)]Cl were prepared [L= deprotonated glycine (Gly), Alanine (Ala), Valine(Val) or Methionine (Met), L- = N-Methylcyclohexyldithiocarbamate(N-MeCHdtc) or Benzyldithiocarbamate(Bzdtc) anion and L= =0,0-Dipropyldithiophosphate (DiPrdtp) or 0,0- Dibenzyldithiophosphate (DiBzdtp) anion. The prepared complexes were characterized by elemental analysis, molar conductivity, IR and UV-Vis spetra, magnatic susceptibility and (1H-nmr) for some of the complexes. The gold(III) complexes were all square planar with the dithiocarbamates or dithiophosphates acted as bidentate ligands coordinated through the two sulfur atoms and the aminoacid anions coordinated through N and O except for the methionine ligand which coordinate through S and N atoms.

Inactivation of Met471Cys tyramine β-monooxygenase results from site-specific cysteic acid formation.

Tyramine β-monooxygenase (TβM), the insect homologue of dopamine β-monooxygenase, is a neuroregulatory enzyme that catalyzes the β-hydroxylation of tyramine to yield octopamine. Mutation of the methionine (Met) ligand to Cu(M) of TβM, Met471Cys, yielded a form of TβM that is catalytically active but susceptible to inactivation during turnover [Hess, C. R., Wu, Z., Ng, A., Gray, E. E., McGuirl, M. M., and Klinman, J. P. (2008) J. Am. Chem. Soc. 130, 11939-11944]. Further, although the wild-type (WT) enzyme undergoes coordination of Met471 to Cu(M) in its reduced form, the generation of Met471Cys almost completely eliminates this interaction [Hess, C. R., Klinman, J. P., and Blackburn, N. J. (2010) J. Biol. Inorg. Chem. 15, 1195-1207]. The aim of this study is to identify the chemical consequence of the poor ability of Cys to coordinate Cu(M). We show that Met471Cys TβM is ~5-fold more susceptible to inactivation than the WT enzyme in the presence of the cosubstrate/reductant ascorbate and that this process is not facilitated by the substrate tyramine. The resulting 50-fold smaller ratio for turnover to inactivation in the case of Met471Cys prevents full turnover of the substrate under all conditions examined. Liquid chromatography-tandem mass spectrometry analysis of proteolytic digests of inactivated Met471Cys TβM leads to the identification of cysteic acid at position 471. While both Met and Cys side chains are expected to be similarly subject to oxidative damage in proteins, the enhanced reactivity of Met471Cys toward solution oxidants in TβM is attributed to its weaker interaction with Cu(I)(M).

Role of Met80 and Tyr67 in the Low-pH Conformational Equilibria of Cytochrome c

The low-pH conformational equilibria of ferric yeast iso-1 cytochrome c (ycc) and its M80A, M80A/Y67H, and M80A/Y67A variants were studied from pH 7 to 2 at low ionic strength through electronic absorption, magnetic circular dichroism, and resonance Raman spectroscopies. For wild-type ycc, the protein structure, axial heme ligands, and spin state of the iron atom convert from the native folded His/Met low-spin (LS) form to a molten globule His/H(2)O high-spin (HS) form and a totally unfolded bis-aquo HS state, in a single cooperative transition with an apparent pK(a) of ~3.0. An analogous cooperative transition occurs for the M80A and M80A/Y67H variants. This is preceded by protonation of heme propionate-7, with a pK(a) of ~4.2, and by an equilibrium between a His/OH(-)-ligated LS and a His/H(2)O-ligated HS conformer, with a pK(a) of ~5.9. In the M80A/Y67A variant, the cooperative low-pH transition is split into two distinct processes because of an increased stability of the molten globule state that is formed at higher pH values than the other species. These data show that removal of the axial methionine ligand does not significantly alter the mechanism of acidic unfolding and the ranges of stability of low-pH conformers. Instead, removal of a hydrogen bonding partner at position 67 increases the stability of the molten globule and renders cytochrome c more susceptible to acid unfolding. This underlines the key role played by Tyr67 in stabilizing the three-dimensional structure of cytochrome c by means of the hydrogen bonding network connecting the Ω loops formed by residues 71-85 and 40-57.

Dynamics of Methionine Ligand Rebinding in Cytochrome c

Geminate recombination of the methionine ligand to the heme iron in ferrous cytochrome c protein following photodissociation displays rich kinetics. It is of particular interest to develop an understanding of fast and slow rebinding time scales, observed in experimental studies, in terms of features of the underlying complex energy landscape. The classical empirical force field in the heme pocket has been extended by incorporating ab initio potential energy surface calculations representing the ground singlet state and quintet state associated with methionine bond breaking and rebinding. An algorithm based on the Landau-Zener nonadiabatic transition theory has been employed to model the electronic surface hopping between two spin states during the process of ligand dissociation and recombination. Multiple conformational substates of the dissociated methionine ligand are found to participate in the reaction dynamics. Varying time scales for interconversion between substates lead to a mechanism elucidating the fast and slow rebinding time scales. The reaction system may be understood in terms of a two-dimensional reaction coordinate distinctly separated from the coupled bath of surrounding protein and solvent degrees of freedom. Insights into the reaction dynamics provided by this study lead to suggestions for future experiments to further probe the role of dynamic heterogeneity in the kinetics of ligand-protein binding.