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The synthesis and self-assembly behavior of biohybrid ABC triblock copolymers consisting of a synthetic diblock, polystyrene-b-polyethylene glycol, where m is varied, and a hemeprotein myoglobin (Mb) or horse radish peroxidase (HRP) are reported. It is revealed that the synthetic diblock copolymer is first functionalized with heme cofactor and then reconstituted with the apoprotein or the apoenzyme to give the protein-containing ABC triblock copolymer.

Combining quantum and molecular mechanics (QM/MM) methods and protein structure prediction algorithms, helix and loop movements were computed along the pathway of CO dissociation from myoglobin (Mb). The principal effect of CO dissociation is shown to be a concerted rotation of the E and F halices, which hold the heme like a clamshell.

The applicability of Badger's rule to heme and non-heme iron-oxygen bonds is examined to gain insights into the protonation state of enzymatic ferryl species. The long Fe-O bonds reported in the crystal structures of the ferryl forms of myoglobin, cytochrome c peroxidase and catalase deviate substantially from the values predicted by Badger's rule, while the short Fe-O bonds obtained from X-ray absorption measurements are in good agreement with Badger's rule.

The selectivity of electrochemical nitric oxide (NO) reduction by heme-didodecyldimethylammonium bromide (DDAB) and myoglobin-DDAB films as a function of potential and pH are examined. The selectivity of the pathway depends on whether a proton or NO molecule is transferred to a Fe(super II)-NO(super -) reaction intermediate and is influenced by pH, NO concentration and potential.

Pulse radiolysis is used to study the kinetics and mechanism of the reaction of oxymyoblobin (MbFeO2) with NO2. The hemeproteins can be instrumental in detoxifying NO2 and hence in pre-empting deleterious processes, such as nitration of proteins.

It is for the first time that orientational probability distribution function for CO transiently docked in myoglobin (Mb) and Hb is established. The ordered protein structure around the active site of the enzyme has a selectivity even for small ligands.

Mononuclear, dinuclear and tetranuclear artificial metalloproteases are prepared by attaching respective catalytic modules containing the Cu(II) complex of cyclen to a derivative of cross-linked polystyrene. The polymeric artificial metalloproteases effectively cleaved peptide bonds of myoglobin by hydrolysis.

Cross-linking of myoglobin (Mb) promoted by 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide within films of polystyrene sulfonate provided remarkable stabilization. Both the polyion film environment and cross-linking seem to play roles in stabilizing protein secondary structure and function at low pH.

The genetic incorporation of a photoreactive unnatural amino acid, p-(2-tetrazole)phenylalanine (p-Tpa), into myoglobin site-specifically in Escherichia coli is described by evolving an orthogonal tTNA/aminoacyl-tRNA synthetase pair and the use of p-Tpa as a bioorthogonal chemical ' handle' for fluorescent labeling of p-Tpa-encoded myoglobin by the photoclick reaction. The structural basis for the biosynthetic incorporation of p-Tpa into proteins is analyzed by solving the X-ray structure of p-Tpa-specific aminoacyl-tRNA synthetase in complex with p-Tpa.

A conserved 2-His-1-Glu metal center, which is found in natural nonheme iron-containing enzymes, is engineered into sperm whale myoglobin by replacing by replacing Leu29 and Phe43 with Glu and His. The analysis has presented a novel protein model of NOR and has provided insights into a newly discovered member of the NOR family, gNOR.

Backbone-modified phosphorothioate DNA and biotin- and maleimide-containing bifunctional linkers are used to achieve controlled alignment of streptavidin (STV), myoglobin, and nanoparticles with nanometer resolution. The proposed method which facilitates the high yield, nanoscale-precision conjugation of multiple proteins to DNA could be used to design a linker to conjugate any proteins and nanomaterials specifically for a wide range of applications.

A strategy is described by which reactive binding of a weakly bound, 'dynamically docked (DD)', complex without a known structure is strengthened electrostatically through optimized placement of surface charges, and its utility in modulating complex formation between myoglobin (Mb) and cytochrome [b.sub.5] ([b.sub.5]) is described. The measurements have shown that the presence of fast photoinduced singlet-electron transfer (ET) across the protein-protein interface.

Strongly electron-withdrawing perfluoromethyl (C[F.sub.3]) groups are substituted as heme side chains of myoglobin (Mb) to obtain larger alterations of the heme electron structure in order to analyze the relationship between the [O.sub.2] affinity of Mb and the electronic properties of heme peripheral side chains. The introduction of electron-withdrawing groups has increased the preferential binding of CO to the protein over that of [O.sub.2].

The effects of solvation on proteins dynamics are examined by using molecular dynamics simulations and myoglobin as a model system. The studies have shown that water is able to interact with proteins in diverse ways, which has led to different kinds of perturbations in their intrinsic dynamic behavior.

A study was conducted to examine the role of copper in regulation of ligand binding in [Cu.sub.B]Mb, a mutant of sperm whale myoglobin as a model for cytochrome c oxidase (CcO). The data demonstrated that the [Cu.sub.B]Mb/[Cu.sup.+] complex reproduces essential structural and kinetic features of CcO very much similar to the complex in Thermus thermophilus CcO (TtCcO) than to bovine CcO (bCcO).

Several studies are conducted to explain the network of pathways involved in the diffusion of the CO molecule inside the myoglobin protein. The entire binding process is shown to be highly dependent and affected by the various cavities present inside the protein.

The distal pocket histidine residue (His64) of horse heart met[Mb.sup.III] (where Mb = myoglobin) has significant effects on the mode of nitrite ion coordination to the iron center. The effects of the mutations on reactivities of the met[Mb.sup.III]s with cysteine as a reducing agent and of the (ferrous) [Mb.sup.II]s with nitrite ion under anaerobic conditions are analyzed.

Myoglobin (Mb) and horseradish peroxidase (HRP) are both reconstituted with a meso-unsubstituted iron corrole and their electronic configurations and peroxidase activities are examined. The catalytic activities of both proteins toward guaiacol oxidation are different and this finding is attributed to the difference in the oxidation states of the corrole iron when these proteins are in the resting state.

Horse myoglobin is used as a model system for assessing suitability of amide hydrogen/deuterium exchange (HDX)- mass spectrometry (MS) with top-down electron capture dissociation (ECD) for analyzing structural transitions of the 17 kDa protein. The results have shown that the level of structural information obtainable with top-down ECD for small to medium-sized proteins have surpassed the traditional HDX-MS experiments, while at the same time has reduced the undesirable amide back exchange.

The studies have shown that water is detected within the distal cavities of myoglobin mutants by subtle changes in the absorbance spectrum of pentacoordinate heme, even when the presence of solvent is not observed in the corresponding crystal structures. The spectral perturbation of pentacoordinate heme has offered a robust system for measuring nonspecific hydration of the active sites of heme proteins.

The studies have shown that the 221 [cm.sup.-1] mode of the photoproduct of the inactive P420 form of inducible nitric oxide synthase ([iNOS.subP420]) has not exhibited any [H.sub.2]O-[D.sub.2]O solvent isotope shift such as that found in the iron-histidine stretching mode of myoglobin, indicating that the proximal ligand of [iNOS.sup.P420] is not a histidine. The studies have supported the scenario that [iNOS.sub.P420] is inactivated by protonation of the native proximal thiolate ligand to a neutral thiol, instead of by ligand switching to a histidine.

Several molecular dynamics simulation studies are conducted to explain the breathing motions of myoglobin, a respiratory protein. The various key residues that work as switches between cavities are also identified.

A systematic global mapping of water motions in the hydration layer around a model protein of apomyoglobin in both native and molten globule states is reported. The results provide insight into a series of correlations of hydrating water dynamics and coupled fluctuations with local protein's chemical and structural properties which are significant for various water behaviors in the hydration layer with wide heterogeneity.

A myoglobin (Mb) triple mutant (D44K/D60K/E85K, Mb(+6)) substituted with Zn-deuteroporphyrin is prepared and cytochrome [b.sub.5] (cyt [b.sub.5]) binding and electron transfer (ET) quenching of the [super 3]ZnMb(+6) triplet state is monitored. The mutations have created the conversion from 'Dynamic Docking' (DD) to simple docking 'simple docking' (SD) energy landscapes.

The deformation of polymersome is examined by loading polymerosomes with dye-labeled ferritin and P[Zn.sub.2]. The studies have shown synergy between ferritin and P[Zn.sub.2], which appears to be a general protein-polymer membrane phenomenon, as replacement of ferritin with bovine serum albumin or equine skeletal myoglobin has resulted in vesicles with similar asymmetric morphology.

CO escaping pathways of Myoglobin are examined in terms of three-dimensional (3D) distribution function that is calculated by the 3D-RISM theory. The partial molar volume changes through the CO escaping pathways have agreed with those from the experiment.

The nature of the Fe-[O.sub.2] bonding in oxy-myoglobin is examined by hybrid quantum mechanical/molecular mechanical calculations and gas-phase calculations using density functional theory (DFT). The protein has strengthened the [Fe.sup.III]-[O.sub.2.sup.-] character of the complex, thus affecting its bonding features and the oxygen binding affinity of Mb.

The experimental and computational studies are carried out to obtain more direct vibrational signatures for protonation of the monooxygen ligand. The results establish the high sensitivity of the Fe-O stretching frequency and FeO tilting which would provide vibrational signatures for the protonation state in structural studies of ferryl heme protein intermediates.

An effective dioxygen activation system in myoglobin having a flavohemin is constructed. The result demonstrates that the replacement of the native hemin with a chemically modified one having a functional group is useful for creating a new function in hemoproteins.

An effort is made to show that it is possible to acquire high-resolution, solution NMR measurements of sol-gel (SG)-encapsulated proteins and also that extensive studies are possible with labeled proteins. The combination of SG-encapsulation and NMR spectroscopy has provided a structural data on intermediates formed by ligand photolysis and snapshots of conformation changes during ligand rebinding.

Studies reveal a new approach to deduce the presence of efficient entry and escape channels in horseradish peroxidase (HRP) with the help of diatomic probe molecules as compared to the myoglobin (Mb). The results show that slower and circuitous escape pathways are developed by this process, as Mb is supposed to be involved in the trapping of diatomic molecules and also shows that the heme relaxation dynamics are not the source of the NO rebinding to Mb.

A resonance Raman (RR) and X-ray absorption characterization of Mb-HNO, which yields new information on bonding within this species, is discussed. The results provided definitive evidence for the structure of Mb-HNO, which supports the structure deduced on the basis of NMR experiments and is the first structural characterization of an HNO bound to a heme protein.

New constituted myoglobins possessing a hydrophobic domain at the terminal of the two heme propionate side chains are constructed. The artificially created domain worked as a barrier against exogenous ligand penetration into the heme pocket, whereas the bound O2 was stabilized in the reconstituted myoglobin as well as in the native one.

A new myoglobin having a suitable amino acid configuration on the heme distal side and a hydrophobic substrate binding suite near the heme pocket at the same time is proposed. The findings indicate that the Km values significantly decrease and the Kcat/km values remarkably increase for 2-methoxyphenol oxidation catalyzed by myoglobins that are prepared by the appropriate hybridization between a mutant and a modified heme and in this approach.

The initial step of the photodissociation pathway of CO-ligated hemoglobin and myoglobin is investigated by means of time-dependent density function theory (TDDFT)(super 6) implemented in the QChem package of programs. It is observed that the iron prefers to occupy the in-plane position in the singlet states.