Nature of a H2O Molecule Confined in the Hydrophobic Interface between the Heme and G-Quartet Planes in a Heme-DNA Complex.

Heme binds selectively to the 3'-terminal G-quartet of all parallel G-quadruplex DNAs to form stable heme-DNA complexes. Interestingly, the heme-DNA complexes exhibit various spectroscopic and functional properties similar to those of hemoproteins. Since the nature of the axial ligands is crucial in determining the physicochemical properties of heme, identification and characterization of the axial ligands in a heme-DNA complex are essential to elucidate the structure-function relationship in the complex. NMR studies of a complex possessing a low-spin ferric heme with a water molecule (H2O) and cyanide ion (CN-) as the axial ligands allowed detailed characterization of the physicochemical nature of the axial H2O ligand. We found that the in-plane asymmetry of the heme electronic structure of the complex is not largely affected by the axial H2O coordination, indicating that the H2O confined in the hydrophobic interface between the heme and G-quartet planes of the complex rotates about the coordination bond with respect to the heme. The effect of the hydrogen(H)/deuterium(D) isotope replacement of the axial H2O on the heme electronic structure was manifested in the isotope shifts of paramagnetically shifted heme methyl proton signals of the complex in such a manner that three resolved peaks associated with axial H2O, HDO, and D2O were observed for each of the heme methyl proton signals. These findings provide not only the basis for an understanding of the nature of the unique axial H2O but also an insight into the molecular mechanism responsible for the control of the heme reactivity in the heme-DNA complex.

Effects of Heme Electronic Structure and Local Heme Environment on Catalytic Activity of a Peroxidase-Mimicking Heme-DNAzyme.

The catalytic cycle of a peroxidase-mimicking heme-DNAzyme involves an iron(IV)oxo porphyrin π-cation radical intermediate known as compound I formed through heterolytic O-O bond cleavage of an Fe3+-bound hydroperoxo ligand (Fe-OOH) in compound 0, like that of a heme enzyme such as horseradish peroxidase (HRP). Peroxidase assaying of complexes composed of chemically modified hemes possessing various electron densities of the heme iron atom (ρFe) and parallel-stranded tetrameric G-quadruplex DNAs of oligonucleotides d(TTAGGG), d(TTAGGGT), and d(TTAGGGA) was performed to elucidate the effects of the heme electronic structure and local heme environment on the catalytic activity of the heme-DNAzyme. The study revealed that the DNAzyme activity is enhanced through an increase in the ρFe and general base catalysis of the adenine base adjacent to the heme, which are reminiscent of the "push" and "pull" mechanisms in the catalytic cycle of HRP, respectively, and that the activity of the heme-DNAzyme can be independently controlled through the heme electronic structure and local heme environment. These findings allow a deeper understanding of the structure-function relationship of the peroxidase-mimicking heme-DNAzyme.

Effect of the Electron Density of the Heme Fe Atom on the Nature of Fe-O2 Bonding in Oxy Myoglobin.

Mössbauer spectroscopy has been used to characterize oxygenated myoglobins (oxy Mbs) reconstituted with native and chemically modified 57Fe-enriched heme cofactors with different electron densities of the heme Fe atom (ρFe) and to elucidate the effect of a change in the ρFe on the nature of the bond between heme Fe and oxygen (O2), i.e., the Fe-O2 bond, in the protein. Quadrupole splitting (ΔEQ) was found to decrease with decreasing ρFe, and the observed ρFe-dependent ΔEQ confirmed an increase in the contribution of the ferric-superoxide (Fe3+-O2-) form to the resonance hybrid of the Fe-O2 fragment with decreasing ρFe. These observations explicitly accounted for the lowering of O2 affinity of the protein due to an increase in the O2 dissociation rate and a decrease in the autoxidation reaction rate of oxy Mb through decreasing H+ affinity of the bound ligand with decreasing ρFe. Therefore, the present study demonstrated the mechanism underlying the electronic control of O2 affinity and the autoxidation of the protein through the heme electronic structure. Carbon monoxide (CO) adducts of reconstituted Mbs (CO-Mbs) were similarly characterized, and we found that the resonance between the two canonical forms of the Fe-CO fragment was also affected by a change in ρFe. Thus, the nature of the Fe-ligand bond in the protein was found to be affected by the ρFe.

Deprotection of a benzyl unit induces a 22π aromatic macrocycle of 3-oxypyripentaphyrin(0.1.1.1.0) with strong NIR absorption.

We report aromaticity switching from a 6π pyridine ring to a 22π macrocyclic ring of 3-oxypyripentaphyrin(0.1.1.1.0). This system has potential applications in photodynamic therapy owing to macrocyclic aromaticity being selectively induced by protecting group removal and strong absorption bands produced in the NIR region especially in methanol.

Characterization of Catalytic Activities and Heme Coordination Structures of Heme-DNA Complexes Composed of Some Chemically Modified Hemes and an All Parallel-Stranded Tetrameric G-Quadruplex DNA Formed from d(TTAGGG).

Heme binds selectively to the 3'-terminal G-quartet (G6 G-quartet) of an all parallel-stranded tetrameric G-quadruplex DNA, [d(TTAGGG)]4, to form a heme-DNA complex. Complexes between [d(TTAGGG)]4 and a series of chemically modified hemes possessing a heme Fe atom with a variety of electron densities were characterized in terms of their peroxidase activities to evaluate the effect of a change in the electron density of the heme Fe atom (ρFe) on their activities. The peroxidase activity of a complex decreased with a decreasing ρFe, supporting the idea that the activity of the complex is elicited through a reaction mechanism similar to that of a peroxidase. In the ferrous heme-DNA complex, carbon monoxide (CO) can bind to the heme Fe atom on the side of the heme opposite the G6 G-quartet, and a water molecule (H2O) is coordinated to the Fe atom as another axial ligand, trans to the CO. The stretching frequencies of Fe-bound CO (νCO) and the Fe-C bond (νFe-C) of CO adducts of the heme-DNA complexes were determined to investigate the structural and electronic natures of the axial ligands coordinated to the heme Fe atom. Comparison of the νCO and νFe-C values of the heme-DNA complexes with those of myoglobin (Mb) revealed that the donor strength of the axial ligation trans to the CO in a complex is considerably weaker than that of the proximal histidine in Mb, as expected from the coordination of H2O trans to the CO in the complex.

A Nuclear Resonance Vibrational Spectroscopic Study of Oxy Myoglobins Reconstituted with Chemically Modified Heme Cofactors: Insights into the Fe-O2 Bonding and Internal Dynamics of the Protein.

The molecular mechanism of O2 binding to hemoglobin (Hb) and myoglobin (Mb) is a long-standing issue in the field of bioinorganic and biophysical chemistry. The nature of Fe-O2 bond in oxy Hb and Mb had been extensively investigated by resonance Raman spectroscopy, which assigned the Fe-O2 stretching bands at ∼570 cm-1. However, resonance Raman assignment of the vibrational mode had been elusive due to the spectroscopic selection rule and to the limited information available about the ground-state molecular structure. Thus, nuclear resonance vibrational spectroscopy was applied to oxy Mbs reconstituted with 57Fe-labeled native heme cofactor and two chemically modified ones. This advanced spectroscopy in conjunction with DFT analyses gave new insights into the nature of the Fe-O2 bond of oxy heme by revealing the effect of heme peripheral substitutions on the vibrational dynamics of heme Fe atom, where the main Fe-O2 stretching band of the native protein was characterized at ∼420 cm-1.

Synergistic Effect of Distal Polar Interactions in Myoglobin and Their Structural Consequences.

In the L29F variant of myoglobin (Mb), the coordination of oxygen (O2) to the heme Fe atom is stabilized by favorable electrostatic interactions between the polar Fe-O2 moiety and the multipole of the phenyl ring of the Phe29 side chain (Phe29 interaction), in addition to the well-known hydrogen bond (H-bond) between the Fe-bound O2 and the 64th residue (distal H-bond; Carver, T. E.; Brantley, R. E., Jr.; Singleton, E. W.; Arduini, R. M.; Quillin, M. L.; Phillips, G. N., Jr.; Olson, J. S. J. Biol. Chem. 1992, 267, 14443-14450). The O2 and carbon monoxide (CO) binding properties and autoxidation of the L29F/H64L and L29F/H64Q variants reconstituted with a series of chemically modified heme cofactors were analyzed and then compared with those of native Mb, and the L29F, H64Q, and H64L variants similarly reconstituted with the chemically modified heme cofactors in order to elucidate the relationship between the Phe29 interaction and the distal H-bond that critically contributes to stabilization of Fe-bound O2. We found that the Phe29 interaction and distal H-bond act cooperatively to stabilize the Fe-bound O2 in such a manner that the Phe29 interaction strengthens with increasing strength of the distal H-bond. Comparison of the functional properties between the L29F and H64L variants indicated that the synergistic effect of the two interactions decreases the O2 dissociation and autoxidation rate constants of the protein by factors of ∼1/2000 and ∼1/400, respectively. Although the CO binding properties of the proteins were not greatly affected by the distal polar interactions, their synergistic effects were clearly and sharply manifested in the vibrational frequencies of the Fe-bound C-O stretching of the proteins.

Simulation Study on Complex Conformations of Aß42 Peptides on a GM1 Ganglioside-Containing Lipid Membrane.

Aggregation and complex formation of amyloid beta (Aß) peptides on a neuronal cell membrane is a hallmark of neuro-disturbance diseases. In this work, we performed molecular dynamics (MD) simulations to investigate the initial stage of interactions of multiple Aß42 peptides on a GM1 ganglioside-containing membrane that mimics a micro-domain on the neuronal cell surface. Conformational changes of Aßs due to adhesion on the membrane and subsequent molecular interactions among the Aßs were monitored. It was suggested from results of the two 1.0 µs simulation trials that stable complexes of Aß peptides were not rapidly generated but that a steady binding of two Aßs was gradually formed. Observation of two Aßs that will be a complex with steady binding revealed that one Aß was bound to the membrane surface, while the other was attached to the first one without strong contact with the membrane. The motion of the first one was restricted and its conformational change was limited, with the basic side-chains of Arg5 and Lys28 working as anchors to hold the Aß helix region on the membrane. In contrast, the second one had high flexibility and showed diversity in its conformation. The second Aß can search for an energetically favorable binding position on the first one. A parallel ß-sheet structure was formed between the C-terminal sides of the two Aßs. Ala30 was critically important to lead the stable ß-sheet conformation at the C-terminal hydrophobic domains of Aßs. In the N-terminal sides, helix structures were kept in both Aßs.

Characterization of Heme Orientational Disorder in a Myoglobin Reconstituted with a Trifluoromethyl-Group-Substituted Heme Cofactor.

The orientation of a CF3-substituted heme in sperm whale myoglobin and L29F, H64L, L29F/H64Q, and H64Q variant proteins has been investigated using 19F NMR spectroscopy to elucidate structural factors responsible for the thermodynamic stability of the heme orientational disorder, i.e., the presence of two heme orientations differing by a 180° rotation about the 5-15 meso axis, with respect to the protein moiety. Crystal structure of the met-aquo form of the wild-type myoglobin reconstituted with 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2,12,18-trimethyl-7-trifluoromethylporphyrinatoiron(III), determined at resolution of 1.25 Å, revealed the presence of the heme orientational disorder. Alterations of the salt bridge between the heme 13-propionate and Arg45(CD3) side chains due to the mutations resulted in equilibrium constants of the heme orientational disorder ranging between 0.42 and 1.4. Thus, the heme orientational disorder is affected by the salt bridge associated with the heme 13-propionate side chain, confirming the importance of the salt bridge in the heme binding to the protein.

Utility of heme analogues to intentionally modify heme-globin interactions in myoglobin.

Myoglobin reconstitution with various synthetic heme analogues was reviewed to follow the consequences of modified heme-globin interactions. Utility of dimethyl sulfoxide as the solvent for water-insoluble hemes was emphasized. Proton NMR spectroscopy revealed that loose heme-globin contacts in the heme pocket eventually caused the dynamic heme rotation around the iron-histidine bond. The full rotational rate was estimated to be about 1400 s(-1) at room temperature for 1,4,5,8-tetramethylhemin. The X-ray analysis of the myoglobin containing iron porphine, the smallest heme without any side chains, showed that the original globin fold was well conserved despite the serious disruption of native heme-globin contacts. Comparison between the two myoglobins with static and rotatory prosthetic groups indicated that the oxygen and carbon monoxide binding profiles were almost unaffected by the heme motion. On the other hand, altered tetrapyrrole array of porphyrin dramatically changed the dissociation constant of oxygen from 0.0005 mm Hg of porphycene-myoglobin to ∞ in oxypyriporphyrin-myoglobin. Heme-globin interactions in myoglobin were also monitored with circular dichroism spectroscopy. The observation on several reconstituted protein revealed an unrecognized role of the propionate groups in protoheme. Shortening of heme 6,7-propionates to carboxylates resulted in almost complete disappearance of the positive circular dichroism band in the Soret region. The theoretical analysis suggested that the disappeared circular dichroism band reflected the cancellation effects between different conformers of the carboxyl groups directly attached to heme periphery. The above techniques were proposed to be applicable to other hemoproteins to create new biocatalysts. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

[24]Pentaphyrin(2.1.1.1.1): A Strongly Antiaromatic Pentaphyrin.

[24]Pentaphyrin(2.1.1.1.1) 1 was synthesized by dehydrogenation of dihydropentaphyrin(2.1.1.1.1) 2 as the first example of vinylogous pentaphyrin. Pentaphyrin 1 takes a roughly planar structure and shows strong antiaromatic character, reflecting a 24π-conjugated circuit. In spite of the antiaromatic character and the relatively small circuit, 1 is stable under ambient conditions.

[62]Tetradecaphyrin and Its Mono- and Bis-Zn(II) Complexes.

A coiled structure of meso-pentafluorophenyl-substituted [62]tetradecaphyrin 1 was revealed by X-ray structural analysis. Synthetic protocols were devised to form mono- and bis-Zn(II) complexes, 1 Zn and 1 Zn2 , selectively. The former displayed a trigonal-bipyramidal pentacoordinated Zn(II) ion as a rare case and a cyclic voltammogram exhibiting eleven reversible redox waves. The latter showed a Ci-symmetric structure with modest Hückel aromaticity owing to a 62 π-electronic circuit as the largest aromatic molecule to date.

Conformational Fixation of a Rectangular Antiaromatic [28]Hexaphyrin Using Rationally Installed Peripheral Straps.

A rectangular [28]hexaphyrin bearing outer straps at the long side has been synthesized by SN Ar reaction of [26]hexaphyrin with allyl alcohol, intramolecular olefin metathesis by using Hoveyda-Grubbs second-generation catalyst, and reduction with NaBH4 . The peripheral straps enforce a rectangular conformation for the [28]hexaphyrin, which shows Hückel antiaromatic character, as confirmed by its planar X-ray structure, a strong paratropic ring current, characteristic UV/Vis/NIR absorption features, small electrochemical HOMO-LUMO gap, and very fast S1 decay.

Characterization of Ground State Electron Configurations of High-Spin Quintet Ferrous Heme Iron in Deoxy Myoglobin Reconstituted with Trifluoromethyl Group-Substituted Heme Cofactors.

We introduced trifluoromethyl (CF3) group(s) as heme side chain(s) of sperm whale myoglobin (Mb) in order to characterize the electronic nature of heme Fe(II) in deoxy Mb using 19F NMR spectroscopy. On the basis of the anti-Curie behavior of CF3 signals, we found that the deoxy Mb is in thermal equilibrium between the 5B2, (dxy)2(dxz)(dyz)(dz2)(dx2-y2), and 5E, (dxy)(dxz)2(dyz)(dz2)(dx2-y2), states of the heme Fe(II), i.e., 5B2 ⇆ 5E. Analysis of the curvature in Curie plots has yielded for the first time ΔH and ΔS values of ∼-20 kJ mol-1 and ∼-60 J K-1 mol-1, respectively, for the thermal equilibrium. Thus, the 5E state is slightly dominant over the 5B2 one at 25 °C. These findings provide not only valuable information about the ground state electronic structure of the high-spin heme Fe(II) in deoxy native Mb but also an important clue for elucidating the mechanism responsible for acceleration of the spin-forbidden oxygenation of the protein.

Effects of Heme Electronic Structure and Distal Polar Interaction on Functional and Vibrational Properties of Myoglobin.

We analyzed the oxygen (O2) and carbon monoxide (CO) binding properties, autoxidation reaction rate, and FeO2 and FeCO vibrational frequencies of the H64Q mutant of sperm whale myoglobin (Mb) reconstituted with chemically modified heme cofactors possessing a variety of heme Fe electron densities (ρ(Fe)), and the results were compared with those for the previously studied native [Shibata, T. et al. J. Am. Chem. Soc. 2010, 132, 6091-6098], and H64L [Nishimura, R. et al. Inorg. Chem. 2014, 53, 1091-1099], and L29F [Nishimura, R. et al. Inorg. Chem. 2014, 53, 9156-9165] mutants in order to elucidate the effect of changes in the heme electronic structure and distal polar interaction contributing to stabilization of the Fe-bound ligand on the functional and vibrational properties of the protein. The study revealed that, as in the cases of the previously studied native protein [Shibata, T. et al. Inorg. Chem. 2012, 51, 11955-11960], the O2 affinity and autoxidation reaction rate of the H64Q mutant decreased with a decrease in ρ(Fe), as expected from the effect of a change in ρ(Fe) on the resonance between the Fe(2+)-O2 bond and Fe(3+)-O2(-)-like species in the O2 form, while the CO affinity of the protein is independent of a change in ρ(Fe). We also found that the well-known inverse correlation between the frequencies of Fe-bound CO (ν(CO)) and Fe-C (ν(FeC)) stretching [Li, X.-Y.; Spiro, T. G. J. Am. Chem. Soc. 1988, 110, 6024-6033] is affected differently by changes in ρ(Fe) and the distal polar interaction, indicating that the effects of the two electronic perturbations due to the chemical modification of a heme cofactor and the replacement of nearby amino acid residues on the resonance between the two alternative canonical forms of the FeCO fragment in the protein are slightly different from each other. These findings provide a new insight for deeper understanding of the functional regulation of the protein.

Synthesis of 5,10-bis(Trifluoromethyl) Substituted ß-Octamethylporphyrins and Central-Metal-Dependent Solvolysis of Their meso-Trifluoromethyl Groups.

5,10-Bistrifluoromethyl substituted ß-octamethylporphyrins were synthesized via a scrambling side reaction of a dipyrromethane precursor in the presence of a large excess of trifluoroacetic acid. Compared with the trans-analogs, the cis-analogs of meso-trifluoromethyl ß-octaalkylporphyrin showed more red-shifted absorption bands. These meso-trifluoromethyl derivatives of ß-octaalkylporphyrins underwent smooth metalation, similar to other common porphyrins, however, the corresponding zinc complexes underwent a type of solvolysis, whereby the trifluoromethyl groups were converted into methoxycarbonyl groups by the methanol used as solvent. UV-visible absorption spectra and X-ray crystal structure analyses revealed that the presence of a methoxycarbonyl substituent did not influence the deformation of the molecular framework and its absorption properties; this is because the methoxycarbonyl has a planar and perpendicular geometry, as opposed to the relatively bulky trifluoromethyl substituent.

Characterization of Heme-DNA Complexes Composed of Some Chemically Modified Hemes and Parallel G-Quadruplex DNAs.

Heme {Fe(II)- or Fe(III)-protoporphyrin IX complex [heme(Fe(2+)) or heme(Fe(3+)), respectively]} binds selectively to the 3'-terminal G-quartet of a parallel G-quadruplex DNA formed from a single repeat sequence of the human telomere, d(TTAGGG), through a π-π stacking interaction between the porphyrin moiety of the heme and the G-quartet. The binding affinities of some chemically modified hemes(Fe(3+)) for DNA and the structures of complexes between the modified hemes(Fe(2+)) and DNA, with carbon monoxide (CO) coordinated to the heme Fe atom on the side of the heme opposite the G6 G-quartet, have been characterized to elucidate the interaction between the heme and G-quartet in the complexes through analysis of the effects of the heme modification on the structural properties of the complex. The study revealed that the binding affinities and structures of the complexes were barely affected by the heme modification performed in the study. Such plasticity in the binding of heme to the G-quartet is useful for the versatile design of the complex through heme chemical modification and DNA sequence alteration. Furthermore, exchangeable proton signals exhibiting two-proton intensity were observed at approximately -3.5 ppm in the (1)H nuclear magnetic resonance (NMR) spectra of the CO adducts of the complexes. Through analysis of the NMR results, together with theoretical consideration, we concluded that the heme(Fe(2+)) axial ligand trans to CO in the complex is a water molecule (H2O). Identification of the Fe-bound H2O accommodated between the heme and G-quartet planes in the complex provides new insights into the structure-function relationship of the complex.

A Dominant Factor for Structural Classification of Protein Crystals.

With the increasing number of solved protein crystal structures, much information on protein shape and atom geometry has become available. It is of great interest to know the structural diversity for a single kind of protein. Our preliminary study suggested that multiple crystal structures of a single kind of protein can be classified into several groups from the viewpoint of structural similarity. In order to broadly examine this finding, cluster analysis was applied to the crystal structures of hemoglobin (Hb), myoglobin (Mb), human serum albumin (HSA), hen egg-white lysozyme (HEWL), and human immunodeficiency virus type 1 protease (HIV-1 PR), downloaded from the Protein Data Bank (PDB). As a result of classification by cluster analysis, 146 crystal structures of Hb were separated into five groups. The crystal structures of Mb (n = 284), HEWL (n = 336), HSA (n = 63), and HIV-1 PR (n = 488) were separated into six, five, three, and six groups, respectively. It was found that a major factor causing these structural separations is the space group of crystals and that crystallizing agents have an influence on the crystal structures. Amino acid mutation is a minor factor for the separation because no obvious point mutation making a specific cluster group was observed for the five kinds of proteins. In the classification of Hb and Mb, the species of protein source such as humans, rabbits, and mice is another significant factor. When the difference in amino sequence is large among species, the species of protein source is the primary factor causing cluster separation in the classification of crystal structures.

Structural and computational study on inhibitory compounds for endonuclease activity of influenza virus polymerase.

Seasonal epidemics and occasional pandemics caused by influenza viruses are global threats to humans. Since the efficacy of currently approved drugs is limited by the emerging resistance of the viruses, the development of new antiviral drugs is still demanded. Endonuclease activity, which lies in the influenza polymerase acidic protein N-terminal domain (PA(N)), is a potent target for novel antiviral agents. Here, we report the identification of some novel inhibitors for PA(N) endonuclease activity. The binding mode of one of the inhibitory compounds to PA(N) was investigated in detail by means of X-ray crystal structure analysis and molecular dynamics (MD) simulation. It was observed in the crystal structure that three molecules of the same kind of inhibitor were bound to one PA(N). One of the three molecules is located at the active site and makes a chelation to metal ions. Another molecule is positioned at the space adjacent to the metal-chelated site. The other molecule is located at a site slightly apart from the metal-chelated site, causing a conformational change of Arg124. The last binding site was not observed in previous crystallographic studies. Hence, the stability of inhibitor binding was examined by performing 100-ns MD simulation. During the MD simulation, the three inhibitor molecules fluctuated at the respective binding sites at different amplitudes, while all of the molecules maintained interactions with the protein. Molecular mechanics/generalized Born surface area (MM/GBSA) analysis suggested that the molecule in the last binding site has a higher affinity than the others. Structural information obtained in this study will provide a hint for designing and developing novel potent agents against influenza viruses.

Electronic control of ligand-binding preference of a myoglobin mutant.

The L29F mutant of sperm whale myoglobin (Mb), where the leucine 29 residue was replaced by phenylalanine (Phe), was shown to exhibit remarkably high affinity to oxygen (O2), possibly due to stabilization of the heme Fe atom-bound O2 in the mutant protein through a proposed unique electrostatic interaction with the introduced Phe29, in addition to well-known hydrogen bonding with His64 [Carver, T. E.; Brantley, R. E.; Singleton, E. W.; Arduini, R. M.; Quillin, M. L.; Phillips, G. N., Jr.; Olson, J. S. J. Biol. Chem., 1992, 267, 14443-14450]. We analyzed the O2 and carbon monoxide (CO) binding properties of the L29F mutant protein reconstituted with chemically modified heme cofactors possessing a heme Fe atom with various electron densities, to determine the effect of a change in the electron density of the heme Fe atom (ρ(Fe)) on the O2 versus CO discrimination. The study demonstrated that the preferential binding of O2 over CO by the protein was achieved through increasing ρ(Fe), and the ordinary ligand-binding preference, that is, the preferential binding of CO over O2, by the protein was achieved through decreasing ρ(Fe). Thus, the O2 and CO binding preferences of the L29F mutant protein could be controlled through electronic modulation of intrinsic heme Fe reactivity through a change in ρ(Fe). The present study highlighted the significance of the tuning of the intrinsic heme Fe reactivity through the heme electronic structure in functional regulation of Mb.