Heme axial methionine fluxion in Pseudomonas aeruginosa Asn64Gln cytochrome c551

Heme axial methionine ligands in ferricytochromes c552 from Hydrogenobacter thermophilus (HT) and Nitrosomonas europaea, both members of the cyt c8 family, display fluxional behavior. The ligand motion, proposed to be inversion at sulfur, results in an unusually small range of hyperfine shifts for heme substituents in these proteins. Herein, heme axial Met fluxion is induced in a structurally homologous cytochrome c551 from Pseudomonas aeruginosa (PA) by substituting heme pocket residue Asn64 with Gln. The mutant, PA-N64Q, displays a highly compressed range of heme substituent hyperfine shifts, temperature-dependent heme methyl resonance line broadening, low rhombic magnetic anisotropy, and a magnetic axes orientation consistent with Met orientational averaging. Analysis of NMR properties of PA-N64Q demonstrates that the heme pocket of the mutant resembles that of HT. This result confirms the importance of peripheral interactions and, in particular, residue 64 in determining axial Met orientation and heme electronic structure in proteins in the cyt c8 family.     

Modulation of the ligand-field anisotropy in a series of ferric low-spin cytochrome c mutants derived from Pseudomonas aeruginosa cytochrome c-551 and Nitrosomonas europaea cytochrome c-552: a nuclear magnetic resonance and electron paramagnetic resonance study

Cytochromes of the c type with histidine-methionine (His-Met) heme axial ligation play important roles in electron-transfer reactions and in enzymes. In this work, two series of cytochrome c mutants derived from Pseudomonas aeruginosa (Pa c-551) and from the ammonia-oxidizing bacterium Nitrosomonas europaea (Ne c-552) were engineered and overexpressed. In these proteins, point mutations were induced in a key residue (Asn64) near the Met axial ligand; these mutations have a considerable impact both on heme ligand-field strength and on the Met orientation and dynamics (fluxionality), as judged by low-temperature electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectra. Ne c-552 has a ferric low-spin (S = 1/2) EPR signal characterized by large g anisotropy with g(max) resonance at 3.34; a similar large g(max) value EPR signal is found in the mitochondrial complex III cytochrome c1. In Ne c-552, deletion of Asn64 (NeN64Delta) changes the heme ligand field from more axial to rhombic (small g anisotropy and g(max) at 3.13) and furthermore hinders the Met fluxionality present in the wild-type protein. In Pa c-551 (g(max) at 3.20), replacement of Asn64 with valine (PaN64V) induces a decrease in the axial strain (g(max) at 3.05) and changes the Met configuration. Another set of mutants prepared by insertion (ins) and/or deletion (Delta) of a valine residue adjacent to Asn64, resulting in modifications in the length of the axial Met-donating loop (NeV65Delta, NeG50N/V65Delta, PaN50G/V65ins), did not result in appreciable alterations of the originally weak (Ne c-552) or very weak (Pa c-551) axial field but had an impact on Met orientation, fluxionality, and relaxation dynamics. Comparison of the electronic fingerprints in the overexpressed proteins and their mutants reveals a linear relationship between axial strain and average paramagnetic heme methyl shifts, irrespective of Met orientation or dynamics. Thus, for these His-Met axially coordinated Fe(III), the large g(max) value EPR signal does not represent a special case as is observed for bis-His axially coordinated Fe(III) with the two His planes perpendicular to each other.     

Effects of heme on the thermal stability of mesophilic and thermophilic cytochromes c: comparison between experimental and theoretical results

We have recently proposed a measure of the thermal stability of a protein: the water-entropy gain at 25?°C upon folding normalized by the number of residues, which is calculated using a hybrid of the angle-dependent integral equation theory combined with the multipolar water model and the morphometric approach. A protein with a larger value of the measure is thermally more stable. Here we extend the study to analyses on the effects of heme on the thermal stability of four cytochromes c (PA c(551), PH c(552), HT c(552), and AA c(555)) whose denaturation temperatures are considerably different from one another despite that they share significantly high sequence homology and similar three-dimensional folds. The major conclusions are as follows. For all the four cytochromes c, the thermal stability is largely enhanced by the heme binding in terms of the water entropy. For the holo states, the measure is the largest for AA c(555). However, AA c(555) has the lowest packing efficiency of heme and the apo polypeptide with hololike structure, which is unfavorable for the water entropy. The highest stability of AA c(555) is ascribed primarily to the highest efficiency of side-chain packing of the apo polypeptide itself. We argue for all the four cytochromes c that due to covalent heme linkages, the number of accessible conformations of the denatured state is decreased by the steric hindrance of heme, and the conformational-entropy loss upon folding becomes smaller, leading to an enhancement of the thermal stability. As for the apo state modeled as the native structure whose heme is removed, AA c(555) has a much larger value of the measure than the other three. Overall, the theoretical results are quite consistent with the experimental observations (e.g., at 25?°C the α-helix content of the apo state of AA c(555) is almost equal to that of the holo state while almost all helices are collapsed in the apo states of PA c(551), PH c(552), and HT c(552)).     

Low‐Temperature EPR and Mössbauer Spectroscopy of Two Cytochromes with His–Met Axial Coordination Exhibiting HALS Signals

C-type cytochromes with histidine-methionine (His-Met) iron coordination play important roles in electron-transfer reactions and in enzymes. Low-temperature electron paramagnetic resonance (EPR) spectra of low-spin ferric cytochromes c can be divided into two groups, depending on the spread of g values: the normal rhombic ones with small g anisotropy and g(max) below 3.2, and those featuring large g anisotropy with g(max) between 3.3 and 3.8, also denoted as highly axial low spin (HALS) species. Herein we present the detailed magnetic properties of cytochrome c(553) from Bacillus pasteurii (g(max) 3.36) and cytochrome c(552) from Nitrosomonas europaea (g(max) 3.34) over the pH range 6.2 to 8.2. Besides being structurally very similar, cytochrome c(553) shows the presence of a minor rhombic species at pH 6.2 (6 %), whereas cytochrome c(552) has about 25 % rhombic species over pH 7.5. The detailed M?ssbauer analysis of cytochrome c(552) confirms the presence of these two low-spin ferric species (HALS and rhombic) together with an 8 % ferrous form with parameters comparable to the horse cytochrome c. Both EPR and M?ssbauer data of axial cytochromes c with His-Met iron coordination are consistent with an electronic (d(xy))(2) (d(xz))(2) (d(yz))(1) ground state, which is typical for Type I model hemes.     

Modulation of structure and dynamics by disulfide bond formation in unfolded states

During oxidative folding, the formation of disulfide bonds has profound effects on guiding the protein folding pathway. Until now, comparatively little is known about the changes in the conformational dynamics in folding intermediates of proteins that contain only a subset of their native disulfide bonds. In this comprehensive study, we probe the conformational landscape of non-native states of lysozyme containing a single native disulfide bond utilizing nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), circular dichroism (CD) data, and modeling approaches. The impact on conformational dynamics varies widely depending on the loop size of the single disulfide variants and deviates significantly from random coil predictions for both NMR and SAXS data. From these experiments, we conclude that the introduction of single disulfides spanning a large portion of the polypeptide chain shifts the structure and dynamics of hydrophobic core residues of the protein so that these regions exhibit levels of order comparable to the native state on the nanosecond time scale.     

Modulation of ligand-field parameters by heme ruffling in cytochromes c revealed by EPR spectroscopy

Electron paramagnetic resonance (EPR) spectra of variants of Hydrogenobacter thermophilus cytochrome c(552) (Ht c-552) and Pseudomonas aeruginosa cytochrome c(551) (Pa c-551) are analyzed to determine the effect of heme ruffling on ligand-field parameters. Mutations introduced at positions 13 and 22 in Ht c-552 were previously demonstrated to influence hydrogen bonding in the proximal heme pocket and to tune reduction potential (E(m)) over a range of 80 mV [Michel, L. V.; Ye, T.; Bowman, S. E. J.; Levin, B. D.; Hahn, M. A.; Russell, B. S.; Elliott, S. J.; Bren, K. L. Biochemistry 2007, 46, 11753-11760]. These mutations are shown here to also increase heme ruffling as E(m) decreases. The primary effect on electronic structure of increasing heme ruffling is found to be a decrease in the axial ligand-field term Δ/λ, which is proposed to arise from an increase in the energy of the d(xy) orbital. Mutations at position 7, previously demonstrated to influence heme ruffling in Pa c-551 and Ht c-552, are utilized to test this correlation between molecular and electronic structure. In conclusion, the structure of the proximal heme pocket of cytochromes c is shown to play a role in determining heme conformation and electronic structure.     

Photoionization of Ferrocytochrome c by 248 nm Laser Light and the Observation of the Early Stages of Ferricytochrome c Unfolding in the Nanosecond-to-millisecond Timescale

Photolysis of ferrocytochrome c by 248 nm laser light in aqueous solution at pH 7 generates hydrated electrons (eaq-) by a monophotonic process with quantum yield phi = 0.034. Approximately three-quarters of the eaq- originate from the heme, which is converted from the ferrous to the ferric state in < 100 ns. The conformational changes associated with the change in the redox state of cytochrome c are either not detectable spectrophotometrically or complete in < 100 ns. Also, under conditions where ferrocytochrome c is stable but ferricytochrome c is unfolded (3 M guanidine, pH 7, 40 degrees C), photoionization of ferrocytochrome c generated ferricytochrome c with similar quantum yield. Under these conditions, the lifetime of native ferricytochrome c is 67 microseconds; it decays via two intermediates with lambda max > 410 nm, neither of which is the thermodynamically favored, unfolded form. These species are putatively identified as unfolding intermediates with nonnative iron ligands, similar to those found during folding of ferrocytochrome c. The results suggest that unfolding, like folding, proceeds by intrachain diffusion and ligand exchange.     

Keeping PASE with WEFT: SHWEFT–PASE pulse sequences for 1H NMR spectra of highly paramagnetic molecules

Metalloproteins are a category of biomolecules in which the metal site is usually the locus of activity or function. In many cases, the metal ions are paramagnetic or have accessible paramagnetic states, many of which can be studied using NMR spectroscopy. Extracting useful information from ¹H NMR spectra of highly paramagnetic proteins can be difficult because the paramagnetism leads to large resonance shifts (~400 ppm), extremely broad lines, extreme baseline nonlinearity, and peak shape distortion. It is demonstrated that employing polychromatic and adiabatic shaped pulses in simple pulse sequences, then combining existing sequences, leads to significant spectral improvement for highly paramagnetic proteins. These sequences employ existing technology, with available hardware, and are of short duration to accommodate short nuclear T₁ and T₂. They are shown to display uniform excitation over large spectral widths (~75 kHz), accommodate high repetition rates, produce flat baselines over 75 kHz while maintaining peak shape fidelity, and can be used to reduce spectral dynamic range. High‐spin (S = 5/2) metmyoglobin, a prototypical highly paramagnetic protein, was used as the test molecule. The resulting one‐dimensional (1D) pulse sequences combine shaped pulses with super‐water elimination Fourier transform, which can be further combined with paramagnetic spectroscopy to give shaped pulses with super‐water elimination Fourier transform–paramagnetic spectroscopy. These sequences require, at most, direct current offset correction and minimal phasing. The performance of these sequences in simple ¹H 1D, 1D NOE, and two‐dimensional NOESY experiments is demonstrated for metmyoglobin and Paracoccus denitrificans Co²⁺‐amicyanin (S = 3/2), and employed to make new heme hyperfine resonance assignments for high‐spin metBjFixLH_151–256, the heme sensing domain of Bradyrhizobium japonicum FixL. Copyright © 2013 John Wiley & Sons, Ltd.     

Elucidating connectivity and metal-binding structures of unlabeled paramagnetic complexes by 13C and 1H solid-state NMR under fast magic angle spinning

Characterizing paramagnetic complexes in solids is an essential step toward understanding their molecular functions. However, methodologies to characterize chemical and electronic structures of paramagnetic systems at the molecular level have been notably limited, particularly for noncrystalline solids. We present an approach to obtain connectivities of chemical groups and metal-binding structures for unlabeled paramagnetic complexes by 13C and 1H high-resolution solid-state NMR (SSNMR) using very fast magic angle spinning (VFMAS, spinning speed >or=20 kHz). It is experimentally shown for unlabeled Cu(II)(Ala-Thr) that 2D 13C/1H correlation SSNMR under VFMAS provides the connectivity of chemical groups and assignments for the characterization of unlabeled paramagnetic systems in solids. We demonstrate that on the basis of the assignments provided by the VFMAS approach multiple 13C-metal distances can be simultaneously elucidated by a combination of measurements of 13C anisotropic hyperfine shifts and 13C T1 relaxation due to hyperfine interactions for this peptide-Cu(II) complex. It is also shown that an analysis of 1H anisotropic hyperfine shifts allows for the determination of electron-spin states in Fe(III)-chloroprotoporphyin-IX in solid states.     

NMR spectroscopy can characterize proteins encapsulated in a sol-gel matrix

Proteins encapsulated within sol-gel matrices (SG) have the potential to fill many scientific and technological roles, but these applications are hindered by the limited means of probing possible structural consequences of encapsulation. We here present the first demonstration that it is possible to obtain high-resolution, solution NMR measurements of proteins encapsulated within a SG matrix. With the aim of determining the breadth of this approach, we have encapsulated three paramagnetic proteins with different overall charges: the highly acidic human Fe3+ cytochrome b5 (cyt b5); the highly basic horse heart cytochrome c (cyt c); and the nearly neutral, sperm whale cyanomet-myoglobin. The encapsulated anionic and neutral proteins (cyt b5; myoglobin) undergo essentially free rotation, but show minor conformational perturbations as revealed by shifts of contact-shifted peaks associated with the heme and nearby amino acids.     

Structural model for an alkaline form of ferricytochrome C

An (15)N-enriched sample of the yeast iso-1-ferricytochrome c triple variant (Lys72Ala/Lys79Ala/Cys102Thr) in an alkaline conformation was examined by NMR spectroscopy. The mutations were planned to produce a cytochrome c with a single conformer. Despite suboptimal conditions for the collection of spectra (i.e., pH approximately equal to 11), NMR remains a suitable investigation technique capable of taking advantage of paramagnetism. 76% of amino acids and 49% of protons were assigned successfully. The assignment was in part achieved through standard methods, in part through the identification of groups maintaining the same conformation as in the native protein at pH 7 and, for a few other residues, through a tentative analysis of internuclear distance predictions. Lys73 was assigned as the axial ligand together with His18. In this manner, 838 meaningful NOEs for 108 amino acids, 50 backbone angle constraints, and 203 pseudocontact shifts permitted the convergence of randomly generated structures to a family of conformers with a backbone RMSD of 1.5 +/- 0.2 A. Most of the native cytochrome c conformation is maintained at high pH. The NOE pattern that involves His18 clearly indicates that the proximal side of the protein, including the 20s and 40s loops, remains essentially intact. Structural differences are concentrated in the 70-80 loop, because of the replacement of Met80 by Lys73 as an axial ligand, and in the 50s helix facing that loop; as a consequence, there is increased exposure of the heme group to solvent. Based on several spectral features, we conclude that the folded polypeptide is highly fluxional.     

Native and unfolded cytochrome c--comparison of dynamics using 2D-IR vibrational echo spectroscopy

Unfolded vs native CO-coordinated horse heart cytochrome c (h-cyt c) and a heme axial methionine mutant cyt c552 from Hydrogenobacter thermophilus ( Ht-M61A) are studied by IR absorption spectroscopy and ultrafast 2D-IR vibrational echo spectroscopy of the CO stretching mode. The unfolding is induced by guanidinium hydrochloride (GuHCl). The CO IR absorption spectra for both h-cyt c and Ht-M61A shift to the red as the GuHCl concentration is increased through the concentration region over which unfolding occurs. The spectra for the unfolded state are substantially broader than the spectra for the native proteins. A plot of the CO peak position vs GuHCl concentration produces a sigmoidal curve that overlays the concentration-dependent circular dichroism (CD) data of the CO-coordinated forms of both Ht-M61A and h-cyt c within experimental error. The coincidence of the CO peak shift curve with the CD curves demonstrates that the CO vibrational frequency is sensitive to the structural changes induced by the denaturant. 2D-IR vibrational echo experiments are performed on native Ht-M61A and on the protein in low- and high-concentration GuHCl solutions. The 2D-IR vibrational echo is sensitive to the global protein structural dynamics on time scales from subpicosecond to greater than 100 ps through the change in the shape of the 2D spectrum with time (spectral diffusion). At the high GuHCl concentration (5.1 M), at which Ht-M61A is essentially fully denatured as judged by CD, a very large reduction in dynamics is observed compared to the native protein within the approximately 100 ps time window of the experiment. The results suggest the denatured protein may be in a glassy-like state involving hydrophobic collapse around the heme.     

Ultrafast Heme Dynamics of Ferric Cytochrome c in Different Environments: Electronic,Vibrational, and Conformational Relaxation

The excited‐state dynamics of ferric cytochrome c (Cyt c), an important electron‐transfer heme protein, in acidic to alkaline medium and in its unfolded form are investigated by using femtosecond pump–probe spectroscopy, exciting the heme and Tryptophan (Trp) to understand the electronic, vibrational, and conformational relaxation of the heme. At 390 nm excitation, the electronic relaxation of heme is found to be ≈150 fs at different pH values, increasing to 480 fs in the unfolded form. Multistep vibrational relaxation dynamics of the heme, including fast and slow processes, are observed at pH 7. However, in the unfolded form and at pH 2 and 11, fast phases of vibrational relaxation dominate, revealing the energy dissipation occurring through the covalent bond interaction between the heme and the nearest amino acids. A significant shortening of the excited‐state lifetime of Trp is observed at various pH values at 280 nm excitation due to resonance energy transfer to the heme. The longer time constant (25 ps) observed in the unfolded form is attributed to a complete global conformational relaxation of Cyt c.     

Mapping the cytochrome C folding landscape

The solution to the riddle of how a protein folds is encoded in the conformational energy landscape for the constituent polypeptide. Employing fluorescence energy transfer kinetics, we have mapped the S.cerevisiae iso-1 cytochrome c landscape by monitoring the distance between a C-terminal fluorophore and the heme during folding. Within 1 ms after denaturant dilution to native conditions, unfolded protein molecules have evolved into two distinct and rapidly equilibrating populations: a collection of collapsed structures with an average fluorophore-heme distance (r) of 27 A and a roughly equal population of extended polypeptides with r > 50 A. Molecules with the native fold appear on a time scale regulated by heme ligation events ( approximately 300 ms, pH 7). The experimentally derived landscape for folding has a narrow central funnel with a flat upper rim on which collapsed and extended polypeptides interchange rapidly in a search for the native structure.     

The earliest events in protein folding: a structural requirement for ultrafast folding in cytochrome C

The folding dynamics of reduced cytochrome c (redcyt c) obtained from tuna heart, which contains a tryptophan residue at the site occupied by His33 in horse heart cytochrome c, was studied using nanosecond time-resolved optical rotatory dispersion spectroscopy. As observed previously for horse heart redcyt c, two time regimes were observed for secondary structure formation in tuna redcyt c: a fast (microseconds) and a slow (milliseconds) phase. However, the fast phase of tuna redcyt c folding was much slower and smaller in amplitude than the same phase in horse. The differences in the fast folding phases suggest that for horse heart redcyt c, the conformers that undergo the fastest observed folding have the His18-Fe-His33 heme configuration, which appears to be necessary, but not sufficient, to poise an unfolded chain conformation for fastest folding in redcyt c.     

Reduced and oxidized cytochrome c4 exhibit differences in dynamics

The temperature-dependent dynamics of the fully reduced and fully oxidized forms of Pseudomonas stutzeri cytochrome c4 have been studied by M?ssbauer spectroscopy. Prior to the dynamic analysis, an efficient labelling strategy has been developed for the expression of highly enriched (57)Fe recombinant cytochrome c4. Subsequently, the protein has been purified to apparent homogeneity. M?ssbauer measurements were recorded in the temperature range 77-240 K for both protein forms. A detailed analysis of the high quality spectra is presented. Based on the information obtained from M?ssbauer spectroscopy, similarities and differences between cytochrome c4, cytochrome c and HiPIP are discussed. The obtained results reveal that (a) cytochrome c4 exists in pure low spin electronic configuration in both oxidation states in the temperature range 77-240 K, (b) the heme pocket is more relaxed in cytochrome c4 than in cytochrome c, (c) the reduced cytochrome c4 is the most flexible at low temperatures, and (d) protein specific dynamics are most distinct in the oxidized protein.     

Influence of the distal his in imparting imidazolate character to the proximal his in heme peroxidase: (1)h NMR spectroscopic study of cyanide-inhibited his42-->ala horseradish peroxidase

The functional higher oxidation states of heme peroxidases have been proposed to be stabilized by the significant imidazolate character of the proximal His. This is induced by a "push-pull" combination effect produced by the proximal Asp that abstracts ("pulls") the axial His ring N(delta)H, along with the distal protonated His that contributes ("pushes") a strong hydrogen bond to the distal ligand. The molecular and electronic structure of the distal His mutant of cyanide-inhibited horseradish peroxidase, H42A-HRPCN, has been investigated by NMR. This complex is a valid model for the active site hydrogen-bonding network of HRP compound II. The (1)H and (15)N NMR spectral parameters characterize the relative roles of the distal His42 and proximal Asp247 in imparting imidazolate character to the axial His. 1D/2D spectra reveal a heme pocket molecular structure that is highly conserved in the mutant, except for residues in the immediate proximity of the mutation. This conserved structure, together with the observed dipolar shifts of numerous active site residue protons, allowed a quantitative determination of the orientation and anisotropies of the paramagnetic susceptibility tensor, both of which are only minimally perturbed relative to wild-type HRPCN. The quantitated dipolar shifts allowed the factoring of the hyperfine shifts to reveal that the significant changes in hyperfine shifts for the axial His and ligated (15)N-cyanide result primarily from changes in contact shifts that reflect an approximately one-third reduction in the axial His imidazolate character upon abolishing the distal hydrogen-bond to the ligated cyanide. Significant changes in side chain orientation were found for the distal Arg38, whose terminus reorients to partially fill the void left by the substituted His42 side chain. It is concluded that 1D/2D NMR can quantitate both molecular and electronic structural changes in cyanide-inhibited heme peroxidase and that, while both residues contribute, the proximal Asp247 is more important than the distal His42 in imparting imidazole character to the axial His 170.     

Nonnative interactions in the FF domain folding pathway from an atomic resolution structure of a sparsely populated intermediate: an NMR relaxation dispersion study

Several all-helical single-domain proteins have been shown to fold rapidly (microsecond time scale) to a compact intermediate state and subsequently rearrange more slowly to the native conformation. An understanding of this process has been hindered by difficulties in experimental studies of intermediates in cases where they are both low-populated and only transiently formed. One such example is provided by the on-pathway folding intermediate of the small four-helix bundle FF domain from HYPA/FBP11 that is populated at several percent with a millisecond lifetime at room temperature. Here we have studied the L24A mutant that has been shown previously to form nonnative interactions in the folding transition state. A suite of Carr-Purcell-Meiboom-Gill relaxation dispersion NMR experiments have been used to measure backbone chemical shifts and amide bond vector orientations of the invisible folding intermediate that form the input restraints in calculations of atomic resolution models of its structure. Despite the fact that the intermediate structure has many features that are similar to that of the native state, a set of nonnative contacts is observed that is even more extensive than noted previously for the wild-type (WT) folding intermediate. Such nonnative interactions, which must be broken prior to adoption of the native conformation, explain why the transition from the intermediate state to the native conformer (millisecond time scale) is significantly slower than from the unfolded ensemble to the intermediate and why the L24A mutant folds more slowly than the WT.     

Recognition and modulation of cytochrome c's redox properties using an amphiphilic homopolymer

An amphiphilic homopolymer scaffold has been used to bind to the protein, cytochrome c. This interaction is analyzed using cyclic voltammetry, native gel electrophoresis, UV-visible absorption, and circular dichroism spectroscopy. The polymer binds to cytochrome c with micromolar affinity and the association of polymer with cytochrome c leads to a structural change of the protein. This conformational change exposes the heme unit of the protein, which affords an opportunity to reversibly modulate its electron-transfer properties. We have also shown that the electrostatic binding of polymer to cytochrome c can be used to disrupt its interaction with its natural partner, cytochrome c peroxidase.     

Influence of amino acid side chain packing on Fe-methionine coordination in thermostable cytochrome C

Paramagnetic NMR and optical studies of the oxidized forms of mesophile Pseudomonas aeruginosa cytochrome c(551) and its quintuple mutant (F7A/V13M/F34Y/E43Y/V78I), and thermophile Hydrogenobacter thermophilus cytochrome c(552) demonstrated that the amino acid side chain packings in the protein interior influence the coordination bond between the heme iron and the axial methionine in the proteins. The strength of heme axial coordinations was found to correlate with the overall protein thermostability.