Functional Implication Guided by Structure-Based Study on Catalase - Peroxidase (KatG) from Haloarcula Marismortui

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Introduction
The catalase-peroxidase (KatG; EC 1.11.1.7)has a variety of roles in different organisms, which distributes widely among fungi, bacteria and archaea.However, the protein sequences of all procaryotic KatGs belong to a member of the class I peroxidase superfamily involving Cytochrome c peroxidase (CcP) and Ascorbate peroxidase (APX) (Welinder, 1991(Welinder, , 1992)).As indicated by the enzyme name, KatG is a bi-functional enzyme exhibiting both catalase and peroxidase activities to prevent potential damage to cellular components by exogenous hydrogen peroxide (H 2 O 2 ) and its deprotonation product.KatG is also interested in its involvement of the activation of anti-tuberculotic pro-drug isonicotinic acid hydrazide (isoniazide, INH) (Bertrand et al., 2004).INH is activated as its peroxidase substrate by KatG from Mycobacterium tuberculosis (Mt).Resulting radical via oxidation (Zhang et al., 1992;Heym et al., 1993) prevents growth of the pathogenic microorganism by inhibiting the synthesis of mycolic acid component of the mycobacterial cell wall.MtKatG shares 55% identity and 69% similarity in its amino acid sequence with KatG from Haroarcula marismortui ( Hm) as homologous protein.Reaction process of catalase and peroxidase activities in the heme-containing hydroperoxidases including class I, class II (fungus lignin peroxidase), class III (classical secretory peroxidase, example being horseradish peroxidase) enzymes and manmalian liver catalase is explained uniformly (Hillar et al., 2000).Ionic (heterolytic) cleavage of the first H 2 O 2 molecule on the heme iron leads to compound I formation, an oxyferryl-radical cation intermediate [Fe IV  =O-Por(･+) or Fe IV =O-Por-aa(･+), aa: side-chain of amino acid of Tyr218(Y218) or Trp95(W95) (all numbering is for HmKatG unless otherwise stated)], is the first step of hydroperoxidase reaction as reaction 1. KatG forms a Por(･+) as well as Y218 and W95 radicals in the absence of reducing substrate (reaction 2; Ivancich et al., 2003).In peroxidases, compound I is reduced in two sequential one-electron transfers, usually from donor (AH, ODA: o-dianisidine), and involve an intermediate called compound II (reactions 3 and 5).Two resonance structures for compound II could exist (reaction 4).

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One of the most common causes of INH resistance is the Ser305Thr (S305T) mutation and, while remaining significant catalase and peroxidase activities, the [S305T] variant enzyme maintains the equivalent affinity for INH (Musser et al., 1996;Haas et al., 1997;Dobner et al., 1997;Marttila et al., 1998;Wengenack et al., 1997;Heym et al., 1995).The MtKatG [S305N]  variant completely lost the ability to convert INH into the InhA inhibitor (Wei et al., 2003).A role for the radicals in activation of the antibiotic is known to proceed by one-electron oxidation producing drug-based radical intermediates (Slayden & Barry III et al., 2000).It indicates that the direct reduction of compound I KatG by INH with pyridine ring is arguing against an activation mechanism requiring amino-acid based radicals.Structural and functional information is available for the crystallographic, kinetics and site-directed mutagenesis studies for HmKatG.The crystal structure of wild-type (WT) HmKatG, [S305T] or [S305A], [R409L], and [M244A] variants is reported otherwise (Sato et al., to be submitted;2011a;2011b;2011c;2011d).Both catalase and peroxidase activities of HmKatG [S305T] have the equal to or lower than those of WT (Sato et al., 2011a;2011b. [S305A] variants exhibit the equal to or lower catalase and the equal to or higher peroxidase activities, respectively, than those of WT (Sato et al., 2011c).[S305A] would yield the prospective complexes with aromatic inhibitor for the higher affinity than that of [S305T].
[R409L] variant, in Arg substitutions of Arg409 to Leu, reveals the higher peroxidase property.These structures in combination with the biochemical characterization of variants lead to identify a few of KatG-specific residues, including the cross-linkage covalent adduct among W95, Y218 and M244, unique to KatGs, G99, Y101, D125, and E194, which is conserved across all KatGs.The mechanistic aspects of KatG catalysis and kinetic studies on site-directed mutagenesis from Hm of equivalent variants from Mt have allowed a fully quantitative rationalization of the catalase reactivity of [S305T] and [R409L] for HmKatG (Sato at al., 2011a).The structure-based computational techniques have provided a mechanistic framework that the binding affinity and rate of turnover for H 2 O 2 of these enzymes may be controlled by bonding (or nonbonding) frontier-orbital due to the presence (or absence) of π-orbital overlap between W95 and Heme.Based on the structures of HmKatG [R409L] variant complexes with (electrophilic reagent) substrate for o-dianisidine (ODA) and (nucleophilic) inhibitor for cyanide anion (CN -), the elevated peroxidase reactivity is gained in binding ODA from reorientation of the side-chain of specific residues in D125, W95 and heme (Sato et al., 2011b).The inhibition of both H 2 O 2 and ODA is observed by such as CN - and salicylhydroxamic acid (2-hydroxybenzohydroxamic acid; SHA) with phenolic ring that are classical heme enzyme inhibitor, respectively, and an inhibitor of compound II KatG with respect to substrate aromatic donor as ODA with dimeric phenolic rings, leading to insight to INH with pyridine ring.SHA and exogenous heme iron ligands compete in binding to the resting enzyme, while phenolic substrates (phenol, p-cresol, and resorcinol) do not competitively inhibit liganding to the heme iron (Modi et al., 1989;Ikeda-Saito et al., 1991).The aromatic substrate binding site on heme iron has been identified by determining the structures of [S305A] and its complexes with SHA.These provide a complete description of substrate binding in KatG (Sato et al., 2011b;2011c R409L] variants are expected to have the higher affinity for SHA and ODA than that of the WT, respectively, and exhibits the equal to or higher peroxidase activity than that of WT. On the other hand, in [R409A], [R409K], and [R409L] variants of other KatGs, the catalase activity decreased while the peroxidase activity did not affected (Carpena et al., 2005;Jakopitsch et al., 2004;Ghiladi et al., 2004).One of the commonest causes of resistance about INH is also the [R409L] mutation in MtKatG and known for peroxidase reaction (Ghiladi et al., 2004;Saint-joanis et al., 1999).It is required for [R409L] equivalent variants from HmKatG to be increasing sensitivity to ODA and, activating significant peroxidase while remaining slightly catalase activities (Sato et al., 2011a;2011b).The single-base mutation between [R (cgg) 409L (ctg)] and [S (agc) 305T (acc)] would be possible from mutational events in natural evolution.It is expected that attempts to generate a "new" function within a laboratory time scale can imitate the entire process used by Nature to evolve and optimize a new function, but these studies illustrate that the acquisition of sequential mutations can produce a continuous pathway for evolution of a "new" function (peroxidase here).
Notably, KatG-specific Met244 sulfur coordination to two carbonyl backbone of Tyr101 (Y101) and Gly99 (G99) and including Tyr218, Trp95 on the distal side of the heme, the unique covalent modification among the side chains of three amino acids (M244, Y218 and W95) are present.It was reported that additionally, phenyl oxygen (Oη) of Y218 and amide nitrogen (N) backbone of M244 in the covalent adduct have hydrogen (H)-bonds with the guanisyl N 1 and N 2 of Arg409 (R409), respectively (Yamada et al., 2002).The covalent adduct has been shown to be generated by autocatalytic process (Ghiladi et al., 2005a).Crystallographic analysis of KatG from Bulkhorderia pseudomallei (Bp) has indicated that the side-chain of R409 (corresponding to HmKatG numbering) shows conformational change depending on both of pH and the physicochemical state of the active site (Carpena et al., 2005).Five amino acid residues, Y101, G99, M244, Y218, and W95 are conserved in all the KatGs but not in the other class I peroxidases.M244L] in BpKatG considerably decreased the catalase activity, whereas the peroxidase activity was even enhanced (Regelsberger et al., 2000;Jakopitsch et al., 2004;Singh et al., 2004 Y218F] in the M244-Y218-W95 covalent adducts induced complete loss of the catalase activity, whereas the peroxidase activity was highly enhanced in Sy KatG (Jakopitsch et al., 2003a;Jakopitsch,et al., 2003b).M244 in the adduct also has any significant role in the enzyme, whereas the substitution of Met to Ile considerably attenuated the catalase activity but did not lose at all, and enhanced the peroxidase activity (Jakopitsch et al., 2004;Singh et al., 2004).Site-specific mutagenesis indicated that the covalent adduct and its relevant was significant to the catalase activity, whereas their effect on the activity was complicated and was hardly explained.Substitution for [M244A] affected the HmKatG structure of the access channel and therefore the enzymatic parameters for the peroxidase activity (Ten-i et al., 2007).This mutation also induced the significant functional change in the active site that would trigger a loss of catalase activity and a high enhancement of peroxidase activity (Sato et al., 2011d).It was also investigated whether the absence of the structural feature unique to KatG, namely M244-Y218-W95 adduct in which are covalently linked together through these side chains, can be correlated with exhibiting the peroxidase reactivity.Resulting in the [M(atg)244A(gca)] substitution, the designed three-base mutation leads to cleavage the covalent bond between M244 and Y218-W95, as was identified in the adducts.It has been constructed the successive triple-base substitution of Met244 to Ala [M244A] and to cleavage the covalent bond amongst M244-Y218-W95 adduct, though this variant would not be expected in natural evolution.Using rounds of site-specific mutagenesis coupled with the structure based-evolution, the unique access will be provided to structural changes that allow enhancement and eventual optimization of the "new" function.
In this paper, the structure-based computational chemical technique can confirm a fully quantitative rationalization of the reactivity mechanism of electron transfer (ET).Interestingly, it was strongly suggested by this structure-base calculation that this variant did not alter compound I formation but can have capacity to restore compound I from compound II, possibly suggesting a yet-to-be defined mechanism for peroxidase that can be allowed to perform in HmKatG., after hydrogen addition to their crystal structures.The residues were confined to the immediate vicinity (surrounded around 3.6Å) among the heme and ligand coordinate of G99 or Y101, M244-Y218-W95 covalent linkages adduct.Geometries were determined by mechanics optimization using augmented MM3.The semi-empirical molecular orbital (MO) method was performed by a MOPAC2002 program / AM1 wavefunction (Stewart, 2002;Dewar et al., 1985).All the sets of molecular orbitals are generated from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO), exhibiting the negative value for MOPAC-specific calculation.When ionization energy (HOMO) is greater than -8 eV including with HOMO density, electrophilic frontier orbital density (fr) and electrophilic superdelocalizability (Sr), the residues have the electrophilic reactivity.

Materials and method
Electron affinity (the LUMO) for nucleophilic group is less than that of -2eV with LUMO density, nuclophilic fr and nuclophilic Sr, exhibiting the reactive indices, fr and Sr to be most reactive (Fukui et al., 1954;1957) with the excitation of electron in the energy gap.An πelectron is transferred from HOMO to LUMO, when HOMO and LUMO polarities are homologous and uniformed, the geometries of the HOMO/LOMO states that have the distance within 3.4Å of van der Waals contact, the matching phases (overlapping the bonding orbitals) and the gap energies within 6eV (Pearson, 1986).It is assumed that the πelectron delocalization of adduct might influence ability of the intrinsic properties of KatG to transfer electron in its conjugate system and then to exhibit the catalase activity (Sato et al., 2011a).For understanding the chemical reactivity and site, the MO calculations were carried out on a PC using BioMedCAChe ver6.1.12.34 (Fujitsu, Tokyo).The outcomes of the in vacuo calculations on coordinate complex, covalent adduct and heme are related to the electronic characteristics of the covalent adducts, SHA, and ODA which bound to heme into the hydrophobic cleft of the variants and its complexes.ODA as a peroxidatic substrate and an electron in the HOMO of the donor molecule as ODA (• + ) cation radical is transferred to the LUMO in the heme of compound I.The reverse is also taking place for SHA.
In addition, when it was difficult for the value of K m (substrate affinity) to measure, conveniently it was quantified by binding energy calculated from the docking study.The binding energy for a given ligand (ΔE ligand ) can be expressed in (eq.7) as the difference in the energy between complex and components (Fukuzawa et al., 2003).
where are the heat of formation energy of each of the three systems, i.e., E ligand of H 2 O 2 , SHA, or ODA and, E enzyme , of the KatG enzyme, and E complex of the enzyme complexes with H 2 O 2 , SHA, or ODA.The binding energy can be estimated to subtract the sum of heat of formation energy values of each system from that of pair of an enzyme and a ligand.
It is indicated that H 2 O 2 molecule leads to compound I formation on the heme iron.In docking exogenous ligands to heme, the binding energy between ligand (H 2 O 2 as an initiator, SHA as an inhibitor of peroxidase compound I and ODA as peroxidase substrate) and enzyme can be approximately estimated from the relative energy difference which subtracts each heat of formation calculated between H 2 O 2 , SHA, ODA and enzyme from that of ligand-enzyme complex.

Results and discussion
3.1 Coordinate, covalent-adduct, heme distal side in the active center and substrate access channel The heme moiety of the [R409L] variant is found to be deeply buried inside the HmKatG and ODA as the peroxidase substrate can access to the active centre only through the narrow cylindrical channel with ~20Å depth from the entrance to the bottom, as shown in Fig1.Side-chain of Asp125 located at the bottom of the channel and showed the structural change in [R409L] complexes with ODA.
As shown in Fig. 2(a), the access channel was composed of LL1 loop, helices B and E, which involved in the active site of R92, H96, and heme.There is three KatG-specific structural characteristics, which consist of first component (π-complex between W95 and Heme), second component (M244-Y218-W95 covalent adduct) and third component (sulfur cation radical centered coordinate complex).One of the unique structural features (of G99, Y101, M244-Y218-W95 covalent adduct and N126) have been revealed in the structures of WT HmKatG, [S305T], [S305A] and [R409L].It consists of the octahedral (six) coordinate sulfur radical cations (>S ￭ + ) center of M244 which is capped at the N-ter end of Helix E by a positively charged amino acid in order to neutralize this helix dipole, ligands among phenyl carbon of (KatG specific) Y218 -W95, carbonyl oxygens of Y101 (KatG specific) and G99 (KatG-typical and conserved with APX) at the C-ter end of the Helix B in KatG.SHA-[S305A] complex shows the phenolic group oxygen of SHA to be of 3.2Å within van der Waals contact with the the -heme edge.In ODA-[R409L] complex, the -heme edge is also confirmed as the primary site oxidation of ODA.(Jakopitsch et al., 2003a;Singh et al., 2004).However, there is a displacement of 2.69Å toward 2.80 Å and dramatic reversal of the dihedral angle χ2 of side D125, which is to bind peroxidase substrate as ODA or water molecule as deriving from H 2 O 2 , respectively.Hence, the mobile D125 residue will also be suggestive of utilizing as either initiator H 2 O 2 or substrate ODA recognition, making it effective in binding substrate, though disruption of π-complexes with heme and W95 known to act on as molecular switch from the catalase to the peroxidase (Carpena et al., 2005).The amino N of backbone I217 forms an H-bond to the oxygen O 1 of carbonyl group of the side D125.Thus no H-atom can be in the C=O 1 group.When O 2 of the D125 can be an ionized carboxyl group at optimum pH6 near pKa value of 4.0, it may be a powerful proton donor for binding the peroxidase substrate with extremely high affinity (Km A = 0.974 μM) for ODA would be attributed to rotate the dihedral angle χ2 by 61.3° of the mobile side D125 in the subunit A of [M244A] variant.
Hence it reorients the sp 2 orbital of the -O 2 (-H) which functions as powerful proton acceptor and has a high affinity for the ODA molecule, when the sp orbital of the -C=O 1 which acts as proton donor and a lower affinity for H 2 O 2 molecule.The role of D125 may form the binding site for the water molecule which derived from the first H 2 O 2 molecule (and then compound I formation), may carry its water molecule out of the active site and may induce and control entry of the second ODA molecule into the cavity.In other words, O 1 with syn lone pairs binds ODA molecule.The one (syn) direction of lone pairs of the carbonyl oxygen (-C=O 1) of side D125 form the more stable H-bind with cation of ODA rather than that of another (anti) direction bound with the amide nitrogen (-NH) of backbone I217 respectively.Thus, it is also indicated by ab initio quantum chemical studies that syn protonation from (-C=O 1) is more favorable to (C-O 2 -H) than anti protonation, implying the syn lone pairs are more basic and therefore bind ODA more readily than do the anti lone pairs (Petersen and Csizmadia, 1979).While the side chain of D125 is rotated around the dihedral angel χ2, since the oxygen orbital changes between (-O 2-H) sp 2 and (=O 1) sp is caused with and without ODA respectively, the ODA is bound to the -meso heme edge by the molecular mechanics and orbital changes of the D125.The D125 has different affinity for ODA from either strong subunit A or weak B, respectively, and facilitates the uptake of ODA in peroxidase, with draining away the water molecule.Therefore, the geometry of side chain of D125 in subunit A allows for higher binding affinity for ODA molecule than that of subunit B, which is consistent with the peroxidase in the [R409L] (Sato,T., et al., 2011b).

Docking and molecular orbital calculations on ODA among WT, S305T and R409L variants
While the difference between [S305T] and [R409L] crystal structures are very subtle changes (with overall root mean square deviation of 0.492 Å) and the structural integrity is highly maintained, the binding energy of such peroxidatic substrate as ODA appears to affect functioning of the enzyme.1. ODA binding affinity against the docking calculation.It is suggested as binding affinity that the value of ∆E ligand is negative to be predicted by docking calculation with the substrate ODA, according to eq.7. ) Only when there are very subtle conformational changes with side-chain of residues and without backbone distortion in the substrate access channel, this structure-based molecular orbital (MO) calculation method appears to provide a useful means of assessing ET pathways and reactivity in heme-containing system.Therefore, an advantage of semi- empirical calculation is the ability to calculate the energies of all the MO of the individual active residues in the protein, which may be expected to be practically much better than no data obtained overtime by ab initio molecular orbital study combined with the large scale computer system of supercomputer.
By examining each subunits of [R409L] structure, the CHB atom is the most reactive site in heme that has electron-withdrawing group, where exhibits as well or greater energy of LUMO, e.g. the eigenvalues of LUMO molecular orbital has -1.43eV and -1.12 eV (Table 2) than those of WT with Subunit A of -1.79 eV and B of -1.96 eV (

Active sites confirmed by structure based on cyanide R409L complex with or without ODA
The peroxidase substrate acts as the electron donor in the peroxidase reaction.In the ODA substrate, the geometry is confined to the biphenyl and binitrogen moieties.The reaction point in ODA (▪ + ) cation radical is also confined to one side of the central part of molecule.
When one electron goes from neutral ODA to the ODA ( + ) positive ion or when changing from the ODA (▪) neutral radical to the ODA (▪ + ) radical-cation states, the ODA molecule undergoes a strain reduction of bicyclic geometry occurring in quinonedimine moiety different from what can be expected from the biphenyl, while the similarity between the central part of the ODA molecule and quinonedimine is maintained, the group of N-C bonds remains planar.The earlier geometry results were obtained from crystallographic structure of complex with ODA.This geometry is consistent with the analysis of the frontier orbitals, confirming that the HOMO level of ODA and LUMO levels of heme are mainly localized over two nitrogen atoms of amino group of the central biphenyl core and CHB atom of the -meso heme edge.The difference in HOMO/LUMO gap energy between 5.7 eV for ODA and 4.9 eV for ODA quinonedimine is the same electronic configurations at different geometries.The lower value of 0.8 eV integrate the information given by the geometry, showing the substantial difference between the strain reduction processes taking place in ODA with respect to binitrogen of quinonedimine and obtained from the results of energy calculations on the basis of AM1 calculations.The frontier orbital approach based on the structure of cyanide [R409L] complexes with ODA also suggests that it is enough to transfer electron from the HOMO ([-7.60eV;-7.72eV]) of ODA cation radical with the electrophilic Sr (0.372, 0.352) to the LUMO ([-1.62 eV; -1.42eV]) of the nucleophilic CHB in heme with nucleophile Sr (0.863, 1.017) greater than that of WT (0.832, 0.912).The HOMO/LUMO difference between ODA cation radical and CHB is of 5.98eV, respectively, and 6.3eV between A and B subunits as shown in Table 3.As suggested by the energy gaps, ET may complete and its cation radical can generate from neutral ODA.
The ODA acts as the cation radical (▪ + ) and is poised for attack by the carbon atom (CHB) of -meso heme edge.The present work also gives evidence for an essential role of CHB as the KatG-typical active site on the peroxidatic activity.The LUMO energy of CHB has the nucleophilic reactivity with and plays a role in binding with ODA of which the HOMO has the electrophilic and forms a positive charge or lone electron pair on N atom in amino groups.Therefore, it is suggested that the peroxidase activity reflects advantageously on the binding affinity.Involvement of the HOMO of the (electrophilic) cationic amino group of ODA in the compound I is oxidized by the CHB of the (nucleophilic) anionic heme group, and an increase in the cavity volume through decay of π-complex between W95 and heme which cause a loss of the catalase reaction, may account for the increased peroxidatic activity and more catalytic efficiency of kcat/Km because of easier binding of ODA than that of WT.The redox intermediate compound I, which has iron oxidized to Fe 4+ =O (oxyferryl species), is formed and a second oxidation equivalent as either a porphyrin cation radical (the LUMO energy of CHB) or the HOMO energy of ODA cation radical, which are phase-matched orbital in respective subunits.
On the other hand, the MO calculation based on structure of cyanide [R409L] is predictive that the unpaired electrons are localized on the iron (IV)-oxo moiety and on W95 by one electron shift of the unpaired electron from W95 to the porphyrin (C1C) near α-meso heme edge.In the absence of ODA, porphyrin cation radical (▪ + ) (LUMO energy for C1C heme of -1.56eV; -1.42eV) has the nucleophilic Sr (0.636, 0.612) greater than that of WT (0.613, 0.583) with the initial formation of anion (-) heme group rather than W95 protein radical (HOMO for N 1 W95 [-7.57eV and -7.54eV]) has formed the electrophilic Sr (0.275, 0.281) less than that of WT (0.285,0.283).The respective energy gap between HOMO and LUMO has 6.01 eV and 6.13eV, ET can be complete occurring within 6eV (Pearson et al., 1986) of the energy gap for the excitation of the electron.The favourably stable neutral radical formation at W95 with concomitant loss of catalase function is followed by due to reduced stabilization of the iron (IV)-oxo containing compound II and increased reversion to compound I when ET in porphyrin ring has been implicated from C1C to CHB.It that a reduction in the cavity volume is suggested through occurrence of π-π* interaction between W95 and heme, which cause a loss of electron with concomitant of reversion to resting state during the catalase reaction, may account for reverting Compound II to Compound I with faster and slower rate of turnover of ODA, respectively, than that of WT in spite of increasing peroxidatic activity and more catalytic efficiency of kcat/Km.Since ODA cation radical (▪ + ) is more erratic and unstable than W95 protein radical, if ODA is able to reach the active site (CHB), the peroxidase reaction occur in close proximity to the CHB after the ferry oxygen abstracts proton from ODA.We have identified a variant, R409L, which provides direct evidence for a bifurcated pathway in which ODA is able to reach the active site (CHB) of heme, and is able to be oxidized by Compound I or Compound II heme intermediates.The presence of substrate is necessary to switch from catalase to peroxidase activities.

MO study based on the structures of S305A complexes with or without SHA
Tripartite architecture is involved in the catalase function, which composed of the first πcomplex between W95 residue and heme, the second covalent adduct of M244-Y218-W95 and the nearly octahedral coordinate complexes among G99, Y101 and M244.As first component stacks of alternating donor and acceptor residues are found in the HmKatG.The relative orientations within the parallel planes of these residues are determined by the energies and charge distributions of the [HOMO] from which an electron will come, and the [LUMO] to which the electron will go.As presented in Table 4, both energies of [E (HOMO)] and [E (LUMO)] were calculated using the AM1 Hamiltonian, of which PM3, PM5 and PM6 wave functions (Stewart, 1989a;1989b;2007) denotes the same tendency.The [HOMO] energy represents the ionization potential of the indole ring in W95, i.e. the [E (HOMO)] of the negative value required to transmit an electron to [E (LUMO)] in heme edge of the porphyrin ring.(In such ET complexes the interplanar spacing of the flat residues is smaller than the normal spacing is of 3.4Å or can be equal to 3.1 Å).It is considered that ET has occurred in such donor-acceptor complexes.The indole ring of W95 in the covalent adduct is almost coplanar with the heme and the "general" ET is likely to occur to the lowest unoccupied heme orbital from the highest occupied π-orbital of W95.When LUMO/HOMO interactions are phase-matched energy gap with small difference between HOMO and LUMO, as well as strong intermolecular interactions, ET can be complete.

Third component (sulfur cation radical formation) in WT HmKatG, [S305A] variants with and without SHA
Since the central S atom in side M244 of third component makes use of six d 2 sp 3 hybrid orbitals, the shape is expected to be octahedral.Octahedral molecule geometries of sulfur coordination complexes have its steric number of 6.When five ligands can be used with a lone electron pair ":" in the octahedral system, an nearly octahedral arrangement complex around the S is created with the five ligands which contains two C atoms in the ethylmethylsulfide group of M244, two O atoms of G99 and Y101, and C 1 atom of the phenol side chain of Y218.The O atom of Y101 and C 1 atom of Y218 takes up an axial position of octahedral SO 2 C 3 ":", whereas one O atom of G99, two C atom of ethylmethylsulfide group and a lone pair electron may take up the equatorial position.
When the central S atom contains more than 6 electrons, the extra electrons form a lone electron pair.If a cation, charged 1+, gathers 6 equivalent ligands around it, S + L 6 , then each of these anions contributes a partial negative charge of -1/6 to the area around the cation, so that local electroneutrality results.For example, if each oxygen atom will experience 1/6 of the charge of the S + ion (=1/6 + ) and S ion will experience 1/6 of an electron (=0.16 -) from each carbon and oxygen atoms, since a sulfur ion S + (0.96+) for WT is surrounded by two carbonyl oxygens and three carbon atoms in G99-C=O (0.47 -) and G101-C=O (0.51 -), C 2(0.48 -) in Y218, C (0.39 -), and C (0.45 -) which each of partial charge was calculated in M244 (SATO et al., 2011a; to be submitted).This value of 1/6 is called a bond valence of surrounding anions, respective atom was added a ground-state orbital slightly larger than the bond valence of 0.16 -or at least 0.2 -, which provides a value for the strength of each interaction between a cation and various anions.The shorter S + •••O interactions would be assumed to be stronger, with a greater partial charge donated by the oxygen atom to the S + ion in order to attain electroneutrality and to maintain the S + Electron must flow from the donor [HOMO] to the acceptor [LUMO], which need to be relatively close (~6eV) in energy (Pearson, 1986).As shown in  .192,0.379;0.336;0.13),nucleophilic Sr (1.263,1.004;3.241,1.141)as well as HOMO density (0.127, 0.217; 0.195, 0.085), and then may fill both roles as HOMO and LUMO by exhibiting the sulfur-ion complexation for MOPAC-specific calculation.In such a coordinate ligand as the carbonyl oxygen atoms of backbone G99 and Y101 plus phenol carbon atom of side Y218, there is also nucleophilic Sr in addition to only HOMO density and electrophilic fr.Thus the third component becomes an excellent source of electron, which an electron was transferred to attractive destination for W95 by way of the second component.Qualitatively, the properties and reactions of indoles have been explained by the resonance theory.π-electrons of an aromatic system is approximated as a linear combination of the functions of suitable contributing LUMO nucleophic phenol coordinate to HOMO electrophilic sulfur center structure and then may produce the electrophilic Sr and positive charge on nitrogen atom in indole group of side W95, reflecting the known electron-donating properties of the indole nitrogen.

First component (π-complex between W95 and heme formation) in S305T, S305A with or without SHA
At first component it was predicted from the MO calculation that a direct ET from W95 to heme and back donation took place during the catalase reaction as a result of a strong electronic interaction between W95 and heme.As shown in Table 2, the W95 π-orbital in [S305T] became both HOMO and LUMO while heme is just the contrary, exhibiting catalase activity.The W95 π-orbital became the HOMO of the complex system in cyanide [R409L] and [S305A], as shown in Tables 2, 3 and 4. The electron orbital is also best explained by the ET from the HOMO of W95 to the LUMO of C1C near α-meso heme edge.Although a transition between W95 orbital and C1C atomic orbital near the point of attachment was a strong contributor to the lower-energy level, when the accepting orbital on the -meso heme edge had no contribution from W95, including the LUMO of CHB as acceptor made a complete preparation for ET from peroxidase substrate, therefore no π-π* interaction has favorably shifted from LUMO energy levels of C1C to CHB, allowing ET to the LUMO of CHB (from the HOMO of ODA as peroxidase substrate to be bound).The CHB is poised for nucleophilic attack by the amino nitrogen lone pair of ODA.The calculated energies between HOMO of ODA and LUMO of CHB show that ET occurs in the [R409L] complex with ODA molecule.The transition from the ground to the first excited state and is mainly described by one-electron excitation from the HOMO of ODA (•+) cation radical to LUMO of CHB.The LUMO ":" of π (i.e.porphyrin ring) is delocalized over the whole C-C bond.The HOMO is located over >C=N (•+) H atom of ODA quinonedimine, consequently the HOMO→LUMO transition implies an ET to aromatic part of π-conjugated system from =NH group.Moreover, these orbitals significantly overlap.The HOMO-LUMO energy gap reveals that they are closely bound and both phase matched therefore become lower in energy.The net effect of ET from the HOMO to LUMO must correspond to the π-π* interaction bonds to be broken in the peroxidase reaction, instead of the -bonds between W95 and heme to be made during the catalase reaction.In [S305A] variant structure, the potential ET is evident from the energies gap between orbital suggestive of [E (HOMO) =-7.55; -7.68 eV] on the indole nitrogen atom (N 1) of W95 involved with nuclophilic Sr (0.331, 0.329) and [E (LUMO) =-1.34; -1.48 eV] on the carbon atom (C1C) electrophilic characteristics associated with nucleophilic superdelocalizability (0.676, 0.675) of the heme in the third component.In contrast, in the SHA-[S305A] complexes it was suggestive of an impossible ET from [E(HOMO)=-7.8;-7.99 eV] on the N 1 to [E(LUMO)=-1.47;-1.47eV]on the C1C due to deficiency in π-π* interaction formation of first component (Pearson RG. 1986).Despite the success of disrupting the π-complexes between heme and W95, one electron will require to be excited by more than 7eV of energy difference from [E (HOMO) =-7.66;-7.69eV] on the (CHB) carbon of heme edge to [E (LUMO) =-0.33;-0.51eV] on the SHA without electrophilic Sr, which is of ODA in ODA- [R409L].It was guided by MO calculation that the LUMO of SHA and the HOMO of CHB of -heme edge cannot act as the electron donor, respectively, and the acceptor.It is indicative of MO results that SHA act as inhibitor, when HOMO/LUMO occurs in reverse from heme to SHA against the order from to heme.

[R409L] structure based molecular orbital study with ODA and CN
It was predicted by [R409L] structure-based calculation that the new band originated as a result of a strong electronic interaction between ODA and -meso carbon (CHB) inheme edge by 5.98-6.3eV than that of N 1 of W95 and C1C near α-meso heme edge by 6.01-6.12eV (Table 3).The former gap energy became more than the latter, increasing the peroxidase and reducing the catalase activities.If SHA serve as hypothetical substrate despite the original inhibitor, a direct ET from SHA to heme would not able to take place during the peroxidase reaction by 7.33-7.18eV(Table 4), since [S305A] cannot have the less gap energy than that of 6.21eV and then exhibits the catalase activity, which added a ground-state π-orbital slightly above the valence bond edge.A transition between SHA orbital and CHB with strong contributions from Fe (3d) atomic orbitals of heme near the point of attachment may also be too strong contributor to generate the new lower-energy band.SHA acts as inhibitor but not substrate, since it has not electrophile Sr which is essential for electorphilic characteristics of ODA to transfer an electron to nucleophilic CHB during peroxidase reaction process.
When the accepting orbital of C1C near the αmeso heme edge, including the LUMO, had no contribution from W95, it can be explained by allowing hole transfer from C1C to CHB in the -heme edge that the CHB π-orbital became the LUMO of the combined system and so ET was complete from ODA to CHB .But the reverse is hardly the case.The interaction has unfavorably changed HOMO and LUMO energy levels, not allowing ET to the LUMO of SHA from the HOMO of the CHB.The calculated HOMO of the CHB and LUMO of the SHA energies show so high that ET cannot occur in the molecule.
The peroxidase reaction has lower activation energy of the phase matched bonding orbital than that of catalase in [R409L].A small HOMO-LUMO energy gap reveals a more reactive compound of peroxidase substrate ligand than that of covalent adduct, which antibonding orbital has out -of -phase interaction and then are higher in energy than bonding orbitals that combine to produce binding interactions.A compound of substrate ligand as ODA with a small HUMO-LUMO energy gap could be considered as a soft base, since the electrophilc Sr of ODA (0.372, 0.352) has also the greater than that of N 1 in W95 (0.275, 0.281) whereas the CHB (0.863, 1.017) exhibits much greater nucleophilic Sr than that of C1C (0.636, 0.612).
This three-part structural feature may generally occur in KatG molecules, over quite long distances from M244 via W95 to CHB at the heme edge, where an electron is localized by disrupting the π-conjugated system between N 1 of W95 and C1C near the heme edge.Then CHB become the LUMO orbital to accept an electron from peroxidase substrate.The energy gap of CHB in the transition-iron-based heme is determined by the π-π* energy difference from property inherent in peroxidase substrate, but cannot be tuned regardless of π-π* splitting between W95 and heme.Therefore, the inhibitor ligand as SHA attached to the transition iron is relatively small.The π-conjugated system of SHA can be made smaller by including ferryl oxygen [Fe 4+ =O] within the conjugated system of heme than that of as ODA.
The ferryl oxygen lowers the excited-state energy owing to a stronger electron affinity relative to carbon.Small π-chromophores such as SHA are used to inhibit resting (π-ground) and compound II (π*-excited) states of heme peroxidase.It involves a direct transition from the π-ground state into the heme, by passing the π*-excited state, which is high in energy.
In the catalase reaction, it would be, for instance, an electron in the HOMO of W95 is transferred to the LUMO of the heme by way of the "hot spot" between heme (C1C) and W95(N 1).In the peroxidase reaction, the HOMO→LUMO transition implies an ET from the HOMO of ODA as a donor molecule to the LUMO of the heme (CHB) as acceptor.An ET prone part of heme and ODA is common in site, where has the large gap and is of opposite phase in LUMO orbital to SHA as inhibitor and the ODAred as product.The pathway is suggested by suppression of ET from the HOMO of CHB to the LUMO of SHA and the ODA red , of course, as the contradiction is untrue.The hydroxamic acid oxygen atom (-NH-OH) of the SHA is thought to be of [-0.33 for subunit A and -0.51 eV for B] as LUMO electron acceptor but in fact acts as HOMO nucleophilic Sr (0.345, 0.358) rather than LUMO electrophilic Sr (0.286, 0.240).The CHB in the -meso carbon and ferryl oxygen por (•+) cation radical is of [-7.66 --7.69eV] which is considered to be electron donor as HOMO nucleophilic Sr (0.860, 0.766).It should give the intermolecular space to the possible ET mechanism of peroxidase reaction that distance between the -NH-OH oxygen atoms of SHA and the nitrogen atom of cyanide heme Fe is shorter than those the ferryl oxygen atom of Fe IV =O of CCP (Itakura et al.,1997) .
The distance between the hydroxamic acid oxygen atom of aromatic donor and the oxygen atom of Fe IV =O is 1.5-1.6Å in KatG, 3.3Å of BHA and 3.4Å of SHA in ARP.These distances are shorter than those of cytochrome C system or CCP (Poulos et al., 1993).Therefore SHA cause to inhibit the KatG.(Ten-i et al., 2007;SATO et al., 2011d).From the docking study, www.intechopen.comhowever, H 2 O 2 binding affinity can be estimated and the proposal space ( §3.1 vide supra in Fig. 3 (A)) among three target residues can be predicted as W95, H96 and D125 from the docking energies which each subunit A and B is of (-30.3kcal/mol;-23.9kcal/mol) for W95, (-29.9 kcal/mol; -18.2 kcal/mol) for H96 and (-32.1 kcal/mol;-30.3kcal/mol)for D125.It is suggested by the structure-based calculation that [M244A] variant has almost the same as affinity for H 2 O 2 between subunit A and B, respectively.

[
A calculated binding energy of subunit A for -76.5 kcal/mol also reveals to have significantly 2-fold higher affinity for inhibitor SHA than that of B for -30.9 kcal/mol.In subunit A, SHA acts as an inhibitor of compound II with respect to a substrate aromatic donor.It is conceivable that SHA behaves as a substrate of compound II but does not imply the competitive binding with donor such as ODA due to binding at the different sites in reactions with compound II intermediate.In the subunit B, SHA bind to the resting state when an ionisable carbonyl group of D125 with pKa value of 4 is not protonated.It is deduced that SHA also inhibits compound I formation by retarding H 2 O 2 binding the iron.
A possible inhibitor binding site has been proposed for target residue D125, in a cavity on the distal side of the -meso heme edge as shown in Fig. 5.As shown in Fig. 4, the proxy end N atom of the amino side chain in substrate ODA is Hbonded with the O 2 atom of D125 in the heme distal pocket.The opposite end N atom forms salt bridge with the side-chain of E194, the carboxyl group of E222 and the carbonyl oxygen atom of backbone S305, with the flexible side chain and without distortion of main chain after binding ODA.The ODA affinity would be increased by the displacement of the side chain of D125 and E194, two active residues on LL1 loop.Oxidation of ODA at nitrogen atom of quinoneimine groups can be also shown by reaction 6 as illustrated in scheme 1.
Indeed the specific enhancements in peroxidise isoenzyme pattern can be influenced by the ODA affinity difference between each subunit.The binding site of another peroxidise substrate as ODA cation radical (ODA (• + ) had been estimated with -46.6 kcal/mol from docking calculations against D125 for subunit A but expected from repulsive energy for subunit B in Table 5.Thus it can be postulated that the active site of subunit A exhibits the higher affinity for the imino (>C=NH) group of ODA (• + ) and deprotonate the amino (-NH 2 ) group of ODA more efficiently than that of the subunit B. The difference of catalytic efficiency in M244A here is of the 18 fold higher subunit A than that of B (Sato et al., 2011d Geometry, which used for the calculations of its model compounds I and II ,was consist of 15 residues which included in R92, W95, H96, G99, T100, Y101, D125, N126, I217, Y218, A244, H259, S305, R409, and heme.In subunit B, the orbital between C1C Heme and N 1 W95 become the binding orbital (in green and yellow) and promotes the bonding of πsystem of indole rings of Trp and pyrole ring of heme, which may be reclaiming compound I.In subunit A, the two atoms between N 1 W95 and C1C Heme is distantly-positioned from 3.9Å, which opposes the π-bonding interaction and cannot transfer electrons, even if each orbital (in red and blue) are attractive and may normally overlap between the bonding molecular orbital.Therefore the reduction of compound II would occur when ODA (• + ) cation radical bound to CHB.
Despite a lack of peroxidase substrate in [M244A] variant, no catalase reactivity against the second H 2 O 2 exhibits at all so that the electron cannot transfer from M244 to the covalent adducts W95 via Y218.In spite of a π-π* electron interaction of the heme with W95 in the covalent adduct, the ability to transfer electron between an electrophile of tyrosinate of Y218 and the nucleophile of sulfur cation was lost by the deletion mutation at the position 244.Therefore KatG is considered the catalase function to use a methionine nucleophile intramolecularly-and octahedrally-coordinated complexes with the carbonyl O atoms of Y101 and G99 as well as known to use tyrosinate-indole electrophiles (Carpena et al., 2005).Crystallographic analysis of HmKatG

Conclusion
Crystal structure of HmKatG has been first determined by 2.0Å resolution and reported the proposal structural characteristics of covalent adduct between S of M244-C 2 of Y218 and C 1 of Y218-Cη2 of W95 in maintaining the catalase activity of the KatG (Yamada et al., 2002).The residues which make up the active site catalytic triad are highly conserved in all KatGs, identified both through functional studies and by comparison with homologues identified in sequenced genomes.X-ray analysis of other KatGs, including Mt, indicates that the corresponding residues are also arranged in a similar catalytic triad.Crystal structure analysis has demonstrated that the Met variant significantly affects W95 in the protein structure on the distal side of the heme since slight changes were observed for the puckering (shrinking and stretching of the interatomic bonds between) heme C1C -N 1 of indole ring as compared to the WT protein (Heering et al., 2002;Santoni et al., 2004).
However, unlike these Met variants from other sources, in the HmKatG [M244A] variant the catalase activity was not detected at all but peroxidase activity was even enhanced.All the reported kinetic constants (rate of turnover, k cat , the affinity of substrate, K m , and catalytic efficiency, k cat /K m ) of otherwise KatG for catalase and peroxidase activities are "apparent" values as conventional Catalase and Peroxidase do not follow typical Michaelis-Mentan kinetics.Kinetic parameters for catalase and peroxidase are determined by fitting the kinetic data to non-linear (mixed) Michaelis-Menten equation and show that isoenzyme pattern of active two catalytic center motifs typical of catalase and peroxidase.That's the reason why KatG is a functional heterodimer in governing KatG dimeric subunits structure.
Crystal structure of the [M244A] variant of HmKatG was of not identical subunits (SATO et al., 2011d).Subunit A disrupted a possible π-interaction between W95 and heme.Including the differences in active site geometry, it would be sufficiently stronger to facilitate the oxoferryl (Fe (IV) =O) reduction in the peroxidase reaction.And back donation of electron from heme edge to W95 would suffice for compound I revitalization in subunit B, suggesting from HOMO/LUMO energy gap and nucleophilic Sr of N 1 based on S305T structure (SATO et al., 2011a).
Characterization of the electron path may identify each site of electrophilic W95 N 1 and nucleophilic heme C1C in HmKatG and influence of heme CHB on peroxidase substrate reduction.Further functional studied with the use of X-ray crystal structure based calculation has given to better understanding of ET pathways and radical formation sites in KatG.This approach was investigated how frontier molecular orbital theory applies to either the inhibitory of a SHA (SATO et al., 2011c; to be submitted) or the reactivity of ODA  compounds (SATO et al., 2011b; to be submitted) with, respectively, single and double phenolic ring.For SHA binding, the side-chain of D125 is rotated around the dihedral angle χ1 by 100.8° whereas ODA binding affinity was enhanced by of 61.3°of D125 residue.On the contrary, in subunit B, the consequent ET from heme to W95 could explain the enhancement of peroxidase and iteratively-generated compound I intermediate.The isoenzyme pattern of peroxidase was discussed in terms of its hetero-dimeric character of peroxidatic subunit A for reduction of compound II and subunit B for reclaiming compound I.The value of catalytic efficiency (k cat /K m ) for the peroxidatic reaction catalyzed by the HmKatG [M244A] variant falls within the expected range for an efficient enzyme (Albery & Knowles, 1976).
The M244-Y218-W95 covalent adduct confirms to be essential for the catalase activity in HmKatG as well as MtKatG (Ghiladi et al., 2005b;2005c).It was also constructed to explore the effect of successive triple-base substitutes for Met244 to Ala and to cleavage the covalent bond amongst the tripeptide.The [M244A] variant that coupled with the structure basedevolution within laboratory time scale is not biochemically associated with INH susceptibility.Despite of INH resistance-conferring variants, this "unnatural" protein engineering for HmKatG, can confirm the inherent catalase functional capability of the C-ter end of E-helix in KatG.Perhaps the most intriguing feature of the MtKatG is its ability to mediate INH susceptibility.In our current working hypothesis for the cause of isoenzyme pattern of peroxidase activity, it is suggest that the drug interacts with the enzyme and is converted by the peroxidase activity into a toxic derivative which acts at a second, as yet unknown, site (Zhang,Y., et al., 1992).
Clearly, further experiments are required to address this hypothesis.For a better understanding of the complex interrelations between catalase and peroxidise, the oxidation of phenols, in the present paper some studies on the kinetic characterization of this enzyme in bacterials has been carried out with the detection of KatG isoenzyme patterns.Whereas C-ter capping by positively charged M244 residues in E-helix is effective to neutralize the helix dipole which lead to destabilization of the helix through entropic effect.Since it may be less effective to neutralize the B-helix dipole, the electron can transfer to carbonyl oxygen atoms of C-ter capping with G99 and Y101 residues in B-helix from heme via the W95-Y218 covalent linkage adduct with M244 centred octahedral coordinate.Just as ODA analogous compounds are bound D125, E194 and E222 on mobile LL1 loop, as the destabilization or cleavage of the covalent adduct linkage between M244 and Y218 may be caused by the mobile Y218 which located on the upstream residues of LL1 loop in binding site.Since the competitive inhibition of ODA substrate may imply in binding SHA inhibitor (as hypothetical INH), the right candidates of the INH may be narrowed down and selected out in a great number of these lead compounds, if the higher binding and lower activation energies for ET during peroxidase reaction are predicted by further docking and additional ab initio MO calculations based on the structure of binding site of INH-sensitive (peroxidase) KatG variants.Peroxidases are highly polymorphic enzymes, and the functionality of each isoenzyme depends on its (acidic) nature and its persistent growth phase of the Mt clinical strains.In order to facilitate the peroxidase activity and to understand the metabolic functions that are needed for the persistence of Mt, compounds that could eradicate persisters effectively can be useful in the design of HIV/anti-tuberculosis drugs.

Fig. 1 .
Fig. 1.Active and ODA Binding Sites of HmKatG.(a; left panel) Space-filling representation of the binding site in showing locations of the five critical residues, Glu194 and Glu222 (blue), Ser305 (green), Asp125 (red) and heme iron (orange).(b; right panel) The vertical cross-sectional view of active site of ODA and CN (gray), surrounded with the residues His96 (cyan), Asp125 and Glu194 (red), Ser305 (yellow) and heme (gray).These figures were constructed using MolFeat v3.0 (FiatLux Corp., Tokyo, Japan).As shown in Fig.3 (A), entering the distal side cavity of [M244A] variant through the constricted portion of the channel, the initiator H 2 O 2 would come into contact with the active-site residues W95 and H96 on Helix B ( §3.6 vide infra in Table5).While the WT, [S305T], [S305A], and [R409L] variants have been stabilized LL1 loop by covalent adduct linkage between Y218 on the mobile LL1 and M244 on Helix E, the [M244A] exhibits the cleavage of the linkage with Y218.When the displacement of E194 and E222 was not endured by the flexible response of the mobile D125 of upstream residues to the downstream portion of LL1 loop, it allows the mouth of the channel to open and to facilitate adequate uptake of substrate into the heme cavity.Side-chains of E194 and E222 on LL1 loop locates at the entrance of the channel and was also affected by this mutation.In subunit A, the side chain D125 disrupted H-bonding interaction with amino nitrogen of backbone I217 in the LL1 loop and the side oxygen O 1 formed H-bond with its own carbonyl oxygen in backbone of D125, resulted perpendicular rotation of χ2 of side D125 to face the imidazole of H96 (Fig.3 (B)) while D125 has been known to be important in the H 2 O 2 oxidation to date(Jakopitsch et al., 2003a;Singh et al., 2004).However, there is a displacement of 2.69Å toward 2.80 Å and dramatic reversal of the dihedral angle χ2 of side D125, which is to bind peroxidase substrate as ODA or water molecule as deriving Fig. 2. (A) Helixes B and E, the 6th Coordinate, the Covalent Adduct and Heme π-complexes in Active Site Structure of WT, and three variants.(B) Structure-based Overlays of the [S305A]-SHA and Cyanide [R409L]-ODA complexes.(a; left panel).The distal R92, H96, N126 on Helix B are shown in blue, W95 on Helix B, Y218 and M244 on Helix E residues in yellow.Heme is shown in purple and orenge sphere represent heme iron atom.The π-π* stacking interaction between W95 and Heme is represented by dashed line between N 1, respectively, and C1C atoms near α-meso heme edge.The H-and coordinated-bonds for the structures of [WT], [S305T], [S305A] and [R409L] are represented by dashed lines (black).(b; right panel).The ligand structures are coincided with -meso heme edge and are avoided overlap considerably between the inhibitor SHA (yellow) and substrate ODA (magenta).The heme is shown in gray, and two residues, W95, H96 and R92, which involved in the binding site for the [S305A] and [R409L], directly superimposed in all cases; for clarity, only one residue is indicated.The coordinated-bonds for the S305A-SHA structure are represented by dashed lines (black).
Fig. 3. (A) Proposed Active Site Structure in [M244A] variant.(B)The mobile D125 in subunit A superposed that of WT. (a; left panel)In Subunit B of [M244A] variant, the distal H96 (on helix B; orange) is shown in blue.The W95 (on helix B), Y218 (on LL1 loop; cyan) and A 244 (on helix E; green) residues are in green, orange, and gray.The heme and its iron atom are represented in magenta sticks and orange sphere.The latent access channel residues of D125, E194 and E222 locate on LL1 loop, showing in red (left panel).(b; right panel) In subunit A of M244A, the Mobile D125 and I217 in pink, distal R92 and H96 are presented in yellow, the Y218 and W95 covalent adduct in cyan.The nitrogen, oxygen and heme iron atoms are colored for dark blue, red, and orange.D125 in subunit B is superimposed in colored blue, white and water molecule is shown as red spheres.The figure is view from the distal side of left panel.The figures were constructed using Pymol (DeLano, 2002).

Fig. 4 .
Fig. 4. The Bind Site Predicted from Docking Calculation with ODA and Confirmed X-ray Structure of Complex.

Fig. 5 .
Fig. 5. Electron Transfer in iron Protoporphyrin IX (heme) and the Adduct or ODA.View showing the stacking of the indole ring of W95 on 3.25Å above pyrol ring C of heme (left panel).The intramolecular ET from porphyrin to the covalent adduct can be explained by the formation π-complexes with W95 ( ▪+ ) and heme, having reverted to compound I from the compound II with no substrate.If ODA is bound to CHB of -heme edge, it allows electron from ODA to the heme to promote oxidation by substrate ODA.Structure of heme and ODA (right panel).This nomenclature is consistent with using in crystallographic work.The figures were constructed using Pymol (DeLano, 2002).
).It is implied for SHA inhibition reaction that the proposal model of INH-MtKatG complex may share structural similarity with SHA in binding site of HmKatG [S305A] variant.Hence, HmKatG [S305T] or [S305A] and [R409L] variants are expressed with an attempt at rational catalytic redesign, to prepare the inhibitor-or substrateenzyme complex, because the [S305A] and [

2.1 Structure based molecular orbital and docking analyses
Functional Implication Guided by Structure-Based Study on Catalase-Peroxidase (KatG) from Haloarcula Marismortui 107 group of heme was H-bonded with either the hydroxyl group of Ser or Thr305, it assumed that it would interfere with the transport of ODA and cause a blockage of the channel.A calculated binding energy in the [R409L] variant (subunit A for -27.6 kcal/mol; B for -73.3 kcal/mol, which is negative and can be discussed by the magnitude of its absolute value as the following from here) also reveals to have significantly high affinity for ODA (Table1) and a possible site has been proposed for [R409L] KatG, in a cavity on the distal side of the heme.Consequently, it is suggested that the porphyrin carbon atom (CHB) of -meso heme edge may serve as a docking site for ODA.Neither catalase nor peroxidase activities may be expected from docking energy (and site) in[S305T].The promising peroxidase despite a lack of catalase activity in [R409L] may result from the localized electronic state of the heme, arising from the H-bonding interaction with ODA.
Each of WT HmKatG and [S305T] variant structures had not exhibited as a potential active site for ODA substrate at all.Since the propionate carbonyl www.intechopen.com

Table 4
The most likely site of binding peroxidase substrate would be the CHB atom in the -meso heme edge where is consistent with ODA recognition site suggested by the docking study in the prospective peroxidase activity.It may be possible to determine the geometries (phases and energies) of the states that would lead to peroxidase activity.It is indicated that the electronic nature of the compound I might be different as could be the mechanism of H 2 O 2 oxidation.Reduction of compound I rather than compound I formation by KatG is the intersection point of the peroxidase and catalase cycle.

Table 2
. The frontier molecular orbital of (a) HOMO and (b) LUMO Energies for Active Site Associated with Catalase and Peroxidase Reactions in WT HmKatG, [S305T] and [R409L] variants.

Table 4 .
Computer-calculated parameters representative of the one-electron oxidation between porphyrin ring of Heme and either indole ring of W95 or aromatic inhibitor of SHA.

M244A] structure based docking and frontier orbital studies with H 2 O 2 , SHA and ODA Both
k cat (the rate of turnover) and K m values (affinity) for H 2 O 2 to [M244A] cannot be detected from kinetic study

Table 5 .
).It has established that subunit B may be correlated with the slow rate of Compound I formation and the fast rate of reduction of Compound II.Subunit A occur the reduction of compound II and can release the product molecule as ODA red resulting from complete reaction of the peroxidase, if subunit A of compound II may adsorb ODA (• + ).Subunit B cannot has the catalase reactivity but regenerate compound I, since its compound II is insensitive to H 2 O 2 and desorbed ODA (• + ).Binding Energies for HmKatG [M244A] Variant Associated with Initiator H 2 O 2 , Inhibitor SHA and Peroxidise Substrate ODA.As shown in Table6, it is supported by the frontier orbital calculation for M244A that catalase activity lost when ET cannot complete between C1C carbon and N 1 nitrogen atom.It is possible to recognize as the peroxidase substrate with the presence of the ODA.However, without the ODA in especially subunit B of M244A, Compound I and W95 were elaborated by ET to W95 ( ▪+ ) cation radical with electrophilc Sr from Por( ▪+ ) via π-complex between them.It is suggested that the C1C coexist with HOMO density, electrophilc fr and electrophilic Sr.When electron donation to the W95( ▪+ ) cation radical is made from ( ▪-)C1C atom on Por ( ▪+ ), compound II reverts to compound I.The working hypothesis of the present study therefore includes the assumption that outcomes of the structure-based calculations can be used as a role to assist reproduction of compound I in peroxidase reaction.