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We review contributions made towards the elucidation of CO and O₂ binding geometries in respiratory proteins. Nuclear magnetic resonance, infrared spectroscopy, Mössbauer spectroscopy, X-ray crystallography and quantum chemistry have all been used to investigate the Fe–ligand interactions. Early experimental results showed linear correlations between ¹⁷O chemical shifts and the infrared stretching frequency (ν_CO) of the CO ligand in carbonmonoxyheme proteins and between the ¹⁷O chemical shift and the ¹³CO shift. These correlations led to early theoretical investigations of the vibrational frequency of carbon monoxide and of the ¹³C and ¹⁷O NMR chemical shifts in the presence of uniform and non-uniform electric fields. Early success in modeling these spectroscopic observables then led to the use of computational methods, in conjunction with experiment, to evaluate ligand-binding geometries in heme proteins. Density functional theory results are described which predict ⁵⁷Fe chemical shifts and Mössbauer electric field gradient tensors, ¹⁷O NMR isotropic chemical shifts, chemical shift tensors and nuclear quadrupole coupling constants (e²qQ/h) as well as ¹³C isotropic chemical shifts and chemical shift tensors in organometallic clusters, heme model metalloporphyrins and in metalloproteins. A principal result is that CO in most heme proteins has an essentially linear and untilted geometry (τ = 4 °, β = 7 °) which is in extremely good agreement with a recently published X-ray synchrotron structure. CO/O₂ discrimination is thus attributable to polar interactions with the distal histidine residue, rather than major Fe–C–O geometric distortions.

Continuous-wave linear-absorption spectroscopy based on THz radiation generated by solid-state photomixers has been applied to the investigation of the dynamics of biomolecules in polyethylene matrices and to line shape studies of HF for diagnostics of semiconductor etching plasmas. The THz spectra of biotin and myoglobin have been obtained using a variable-temperature, cryogenic sampling system. The spectrum of biotin displays a small number of discrete absorptions over the temperature range from 4.2 K to room temperature while the spectrum of myoglobin has no obvious resonance structure at the >10% fractional absorption level. Spectral predictions from the lowest energy ab initio conformations of biotin are in poor agreement with experiment, suggesting the need to include condensed-phase environmental interactions for qualitative predictions of the THz spectrum. Vibrational anharmonicity is used to model the line shapes that result from drastic changes in vibrational sequence level populations of biotin over this temperature range. Anharmonicity factors (χ_eω_e/ω_e) at the levels of 0.1% to 0.8% are obtained from non-linear least squares fits of the observed resonances and illustrate their important for refining model predictions. Application of the photomixer system to line shape studies in etching plasmas has been used to study the formation efficiency and translational temperature of HF at 1.2 THz under different operating conditions. These results will aid in understanding the chemistry of industry-standard fluorocarbon and oxygenated fluorocarbon etching plasmas.

Myosin heavy chain (MHC) is a structurally bound contractile protein of the thick filaments. The magnitude of myoskeletal injury may be estimated by serial determinations of these contractile proteins released into the circulation as a consequence of loss of cell membrane integrity. The polymorphism of MHC is believed to be responsible, at least in part, for the variable contraction velocity of muscles. It was the aim of the present study to confirm the utility of MHC for the assessment of skeletal muscle damage within 24 hr of injury in patients with myoskeletal injuries. Plasma MHC fragments was measured in 25 patients with muscle injuries. Samples were obtained immediately on hospital admission (A1) and 24 hr after admission (A2). These were compared with 15 noninjured controls. In addition, creatine kinase (CK), myoglobin and cardiac troponin I (cTnI) were measured in order to confirm the extent of the injury and to exclude protein released from the heart. Radioimmunoassay, enzyme immunoassay, and one-step immunoenzymometric assay (IEMA) techniques were used for measurements of plasma MHC fragments, CK plus myoglobin and cTnI, respectively. Mean (SD) uU/l of MHC was (94.1±116.1) p=NS and (673.3±1943) p<0.0001 on A1 and A2, respectively, compared to controls (96.8±96.35). CK (IU/l) was 226.0±231.0 on A1 and 451.0±678.0 on A2 compared to 104.0±51.0 on controls. Myoglobin was 200% higher at A1 and A2 than at controls. We substantiate previous study and conclude that MHC could be a useful tool in the study of myoskeletal injuries in human.

Many proteins contain cofactors, such as heme, ATP and metal ions. Binding of cofactors is not only essential for their biological functions, but also can reshape the intrinsic energy landscape of protein molecules and modulate the folding and stability. However, the molecular mechanism of cofactor coupled protein folding is not well understood. In this work, we study the cofactor coupled folding of myoglobin, which is a typical cofactor (heme) containing protein, by performing molecular dynamics simulations with a structure-based protein model developed based on the energy landscape theory. We showed that the heme binding increases the stability of the myoglobin. More importantly, the heme binding tends to increase the protein folding cooperativity, and switch the folding process from a “three-state” mechanism to a “two-state” mechanism. We also showed that the folding pathways of the myoglobin can be modulated by the heme binding. By performing comparative simulations, we revealed that the above effects of heme binding are resulted from the heme induced folding of F-helix, which is otherwise unstructured at apo state, and the heme mediated contacting interactions around the heme binding site. The simulation results are consistent with available experimental data, and provide insights into the molecular mechanism of the effects of cofactor binding on the protein folding and stability.

Recent investigations of the His93Gly (H93G) "cavity" mutant of myoglobin as a versatile scaffold for modeling heme states are described. The difference in accessibility of the two sides of the heme in H93G myoglobin makes it possible to generate mixed ligand adducts in the ferric state that are difficult to prepare with heme models in organic solvents. In addition, the protection provided to the heme by the protein environment allows for the preparation of stable oxyferrous and oxo-iron(IV) complexes at near-ambient temperatures with variable ligands trans to the normally reactive dioxygen and oxo substituents. The extensive range of possible complexes that can be generated using the H93G system is illustrated with examples involving imidazole, phenolate, benzoate, thiolate and thiol ligands bound to the proximal side of the heme iron.

The modification of myoglobin is an attractive process not only for understanding its molecular mechanism but also for engineering the protein function. The strategy of myoglobin functionalization can be divided into at least two approaches: site-directed mutagenesis and reconstitution with a non-natural prosthetic group. The former method enables us to mainly modulate the physiological function, while the latter has the advantage of introducing a new function on the protein. Particularly, replacement of the native hemin with an artificially created hemin having hydrophobic moieties at the terminal of the heme-propionate side chains serves as an appropriate substrate-binding site near the heme pocket, and consequently enhances the peroxidase and peroxygenase activities for the reconstituted myoglobin. In addition, the incorporation of the synthetic hemin bearing modified heme-propionates into an appropriate apomyoglobin mutant drastically enhances the peroxidase activity. In contrast, to convert myoglobin into a cytochrome P450 enzyme, a flavin moiety as an electron transfer mediator was introduced at the terminal of the heme-propionate side chain. The flavomyoglobin catalyzes the deformylation of 2-phenylpropanal in the presence of NADH under aerobic conditions through the peroxoanion formation from the oxygenated species. In addition, modification of the heme-propionate side chains has an significant influence on regulating the reactivity of the horseradish peroxidase. Furthermore, the heme-propionate side chain can form a metal binding site with a carboxylate residue in the heme pocket. These studies indicate that modification of the heme-propionate side chains can be a new and effective way to engineer functions for the hemoproteins.

Aspects of protein engineering of cytochrome P450 (P450) and myoglobin (Mb) to construct selective oxygenation catalysts have been described. Heme enzymes are known as biocatalysts for various oxidations but the design of substrate specificity has still remained one of the significant challenges because of dynamic nature of enzyme-substrate interactions. In particular, P450s are the most interesting targets among the heme enzymes because they are able to catalyze many types of monooxygenations such as hydroxylation, epoxidation, and sulfoxidation with high selectivity. Thus, many researchers have made efforts to convert the selectivity for natural substrates into that for unnatural substrates by several protein engineering approaches. On the other hand, we have reported a rational design of Mb to convert its oxygen carrier function into that of peroxidase or peroxygenase. The Mb mutants prepared in our work afford oxo-ferryl porphyrin radical cation (compound I) as observable species in Mb for the first time. Furthermore, some of the mutants we have constructed are useful for enantioselective oxygenations by oxygen transfer from the Mb-compound I to substrates.

Sulfoxylate SO₂H⁻(SO₂²⁻), a strong reducing agent readily produced by hydrolysis of thiourea dioxide, reacts with ferric myoglobin (Mb) to reversibly produce Fe(II)-Mb, starting from either aerobic or anaerobic conditions. Exposure of Fe(II)-Mb to excess sulfoxylate further produces Fe(II)-CO-Mb. Fe(II)-Mb can be regenerated by reoxidation with ferricyanide at this stage; hemin, rubredoxin and cytochrome c show a similar reactivity towards sulfoxylate. The source of CO is not the protein moiety, nor is it the heme or the thiourea dioxide – but rather CO₂, via its reaction with sulfoxylate when the latter is used in large excess. These findings provide a convenient single-step route to carbon monoxide heme adducts, without the need to manipulate toxic CO gas.

Protoheme IX is a typical iron porphyrin cofactor, showing a variety of reactivities in many hemoproteins under the reaction environments provided by protein matrices. Chemical modification of the protoheme cofactor is expected to be a versatile strategy to design hemoproteins possessing unique functions. This review focuses on the conversion of a hemoprotein, mainly myoglobin (an oxygen-storage hemoprotein), into a protein having different functions from the original ones by replacement of the protoheme cofactor with synthetic cofactors. The myoglobin having anionic patches pended to the heme propionates effectively binds electron-accepting proteins or small cationic organic molecules on the protein surface, resulting in enhanced efficiency of the photoinduced electron transfers from the myoglobin to these electron acceptors. Furthermore, the peroxidase and peroxygenase activities are also enhanced due to the facile substrate accesses. The attachment of the chemically active moiety such as flavin at the heme terminal is also important to give P450-like function to the native myoglobin. The employment of a structural isomer of porphyrin as an artificial cofactor gives rise to remarkably high dioxygen affinity and peroxidase activity in myoglobin, and allows us to easily detect high-valent species of the porphyrin isomer in HRP. These examples provide a clear insight into hemoprotein modifications based on synthetic chemistry as well as genetic amino acid mutations.

A non-precious metal catalyst (NPMC) promoting a four-electron oxygen reduction reaction (ORR) was synthesized by heat treatment of myoglobin (Mb) containing a heme (iron protoporphyrin IX) as a source of iron, nitrogen, and carbon atoms. Samples of the mixture of Mb and carbon black (Vulcan XC72R: VC) were pyrolyzed at 740, 840, 940, 1040 or 1140°C under N₂ flow. The microstructures of the carbonized Mb catalysts were characterized by XRD, Raman spectroscopy, XPS, and TEM. Results indicate that the iron-containing active site is embedded within the surface structure in an amorphous domain of the carbon materials. The catalyst ink in a 0.05 wt% Nafion solution in isopropanol was coated onto a glassy carbon electrode and the ORR activity of Mb-based NPMCs was evaluated in a rotating disk electrode experiment in an O₂-saturated 0.1 M HClO₄ solution at 25°C. The catalyst synthesized at 940°C has the highest ORR activity in terms of the onset potential and the current density. In contrast, pyrolytic temperatures above 940°C decrease the activity, suggesting that the active structure of the catalyst apparently decomposes at higher temperatures. The Koutecky–Levich plots indicate that the Mb-based catalyst prepared at 940°C catalyzes four-electron ORR (n = ca. 4). The catalysts prepared at other temperatures have n values of 3.6 at 740°C, 3.7 at 840°C, and 2.9 at 1040°C. The ORR of Mb/VC is diffusion-controlled at potentials lower than 0.3 V (vs. RHE) and the onset potential is 0.84 ± 0.01 V.

If life without heme-Fe were at all possible, it would definitely be different. Indeed this complex and versatile iron-porphyrin macrocycle upon binding to different “globins” yields hemeproteins crucial to sustain a variety of vital functions, generally classified, for convenience, in a limited number of functional families. Over-and-above the array of functions briefly outlined below, the spectacular progress in molecular genetics seen over the last 30 years led to the discovery of many hitherto unknown novel hemeproteins in prokaryotes and eukaryotes. Here, we highlight a few basic aspects of the chemistry of the hemeprotein universe, in particular those that are relevant to the control of heme-Fe reactivity and specialization, as sculpted by a variety of interactions with the protein moiety.

Chlorin e6 (Ce6) and its derivatives are among the most important photosensitizers in photodynamic therapy. Due to their intense fluorescence, chlorins may also be used for diagnostics. However, low solubility in water and high tendency to aggregation restrict their medical use. Here we demonstrate that apo-myoglobin, by reinserting Ce6 in its heme binding pocket, can be used to monomolecularly disperse it. The reconstructed myoglobin-Ce6 adduct presents noticeable changes in the photophysical properties of the chromophore. A red-shift, in particular in the transparency window, can be observed in the absorption and in the emission spectra of the adduct compared to the spectra of the free chlorin in PBS. The adduct presents a higher quantum yield and an increased excited-state lifetime with respect to the free Ce6. The binding of Ce6 to apo-myoglobin determines a decrease of the ¹O₂ generation but a three-fold increase of peroxides production, determining globally an increase in the performance of Ce6 as a photosensitizer and imaging agent.

Nitroorganics present a general concern for a safe environment due to their health hazards. However, some nitroorganics such as metronidazole (Mtz) and chloramphenicol (CAM) also possess medicinal value. Mtz and CAM can undergo reductive bioactivation presumably via their nitroso derivatives. We show, using UV-vis spectroscopy, that sperm whale myoglobin (swMb) and its distal pocket mutants retaining H-bonding capacity react with Mtz in the presence of dithionite to generate products with spectra suggestive of the Fe-bound nitroso (Fe–RNO; ${\lambda }_{max}$$\sim$420 nm) forms. We have crystallized and solved the X-ray crystal structure of an H64Q swMb-acetamide compound to 1.76 Å resolution; the formation of this compound results from the serendipitous crystallographic trapping, by the heme center, of acetamide from the reductive decomposition of Mtz. Only one of the swMb proteins, namely H64Q swMb with a relatively flexible Gln64 residue, reacted with CAM presumably due to the bulky nature of CAM that generally may restrict its access to the heme site.

Novel feather-like CeO₂ microstructures were achieved by a thermal decomposition approach of Ce(OH)CO₃ precursor. The Ce(OH)CO₃ was obtained from a solvothermal method employing Ce(NO₃)₃.6H₂O with C₆H${}_{12}$N₄ and C${}_{16}$H${}_{33}$(CH₃)₃NBr (CTAB) at 190${}^{\circ }$C in a water–PEG-200 mixed solution. The feather-like CeO₂ dendrite was obtained by thermal conversion of the feather-like Ce(OH)CO₃ at 650${}^{\circ }$C in air. A reasonable growth mechanism was proposed with the soft-template effect of PEG-200. The electrochemical behavior and enzyme activity of myoglobin (Mb) immobilized on CeO₂–Nafion modified glassy carbon electrode (GCE) are demonstrated by cyclic voltammetric measurements. The results indicate that CeO₂ can obviously promote the direct electron transfer between the Mb redox centers and the electrode. The Mb on CeO₂–Nafion behaves as an elegant performance on the electrochemical reduction of trichloroacetic acid (TCA) from 0.32$\mu$M to 2.28$\mu$M. The detection limit is estimated to be 0.08$\mu$M.

We model helical polypeptides in an aqueous environment by explicitly evaluating winding probabilities of biopolymers. To account for differences in reaction to the solvent of the various types of amino acids forming the chainlike biopolymer, a length-dependent drift coefficient A(s) is used. As an application, we express A(s) in terms of a Bessel function to generate a sequence of winding and non-winding segments and compare this with the α-helical segments of myoglobin.

Continuous-wave linear-absorption spectroscopy based on THz radiation generated by solid-state photomixers has been applied to the investigation of the dynamics of biomolecules in polyethylene matrices and to line shape studies of HF for diagnostics of semiconductor etching plasmas. The THz spectra of biotin and myoglobin have been obtained using a variable-temperature, cryogenic sampling system. The spectrum of biotin displays a small number of discrete absorptions over the temperature range from 4.2 K to room temperature while the spectrum of myoglobin has no obvious resonance structure at the >10% fractional absorption level. Spectral predictions from the lowest energy ab initio conformations of biotin are in poor agreement with experiment, suggesting the need to include condensed-phase environmental interactions for qualitative predictions of the THz spectrum. Vibrational anharmonicity is used to model the line shapes that result from drastic changes in vibrational sequence level populations of biotin over this temperature range. Anharmonicity factors (χ_eω_e/ω_e) at the levels of 0.1 % to 0.8 % are obtained from non-linear least squares fits of the observed resonances and illustrate their important for refining model predictions. Application of the photomixer system to line shape studies in etching plasmas has been used to study the formation efficiency and translational temperature of HF at 1.2 THz under different operating conditions. These results will aid in understanding the chemistry of industry-standard fluorocarbon and oxygenated fluorocarbon etching plasmas.