柳澤 幸子

Abstract Antimicrobial resistance (AMR) is a global health problem. Despite the enormous efforts made in the last decade, threats from some species, including drug-resistant Neisseria gonorrhoeae, continue to rise and would become untreatable. The development of antibiotics with a different mechanism of action is seriously required. Here, we identified an allosteric inhibitory site buried inside eukaryotic mitochondrial heme-copper oxidases (HCOs), the essential respiratory enzymes for life. The steric conformation around the binding pocket of HCOs is highly conserved among bacteria and eukaryotes, yet the latter has an extra helix. This structural difference in the conserved allostery enabled us to rationally identify bacterial HCO-specific inhibitors: an antibiotic compound against ceftriaxone-resistant Neisseria gonorrhoeae. Molecular dynamics combined with resonance Raman spectroscopy and stopped-flow spectroscopy revealed an allosteric obstruction in the substrate accessing channel as a mechanism of inhibition. Our approach opens fresh avenues in modulating protein functions and broadens our options to overcome AMR.

Low body temperature predicts a poor outcome in patients with heart failure, but the underlying pathological mechanisms and implications are largely unknown. Brown adipose tissue (BAT) was initially characterised as a thermogenic organ, and recent studies have suggested it plays a crucial role in maintaining systemic metabolic health. While these reports suggest a potential link between BAT and heart failure, the potential role of BAT dysfunction in heart failure has not been investigated. Here, we demonstrate that alteration of BAT function contributes to development of heart failure through disorientation in choline metabolism. Thoracic aortic constriction (TAC) or myocardial infarction (MI) reduced the thermogenic capacity of BAT in mice, leading to significant reduction of body temperature with cold exposure. BAT became hypoxic with TAC or MI, and hypoxic stress induced apoptosis of brown adipocytes. Enhancement of BAT function improved thermogenesis and cardiac function in TAC mice. Conversely, systolic function was impaired in a mouse model of genetic BAT dysfunction, in association with a low survival rate after TAC. Metabolomic analysis showed that reduced BAT thermogenesis was associated with elevation of plasma trimethylamine N-oxide (TMAO) levels. Administration of TMAO to mice led to significant reduction of phosphocreatine and ATP levels in cardiac tissue via suppression of mitochondrial complex IV activity. Genetic or pharmacological inhibition of flavin-containing monooxygenase reduced the plasma TMAO level in mice, and improved cardiac dysfunction in animals with left ventricular pressure overload. In patients with dilated cardiomyopathy, body temperature was low along with elevation of plasma choline and TMAO levels. These results suggest that maintenance of BAT homeostasis and reducing TMAO production could be potential next-generation therapies for heart failure.

Disproportionation of Cpd II models depends on the electron-richness of the porphyrin ligand; Cpd II with an electron-deficient ligand is difficult to disproportionate, whereas Cpd II with an electron-rich ligand readily disproportionates to form Cpd I as a true oxidant.

Nitric oxide (NO) reductase from the fungus <italic>Fusarium oxysporum</italic> is a P450-type enzyme (P450nor) that catalyzes the reduction of NO to nitrous oxide (N₂O) in the global nitrogen cycle. In this enzymatic reaction, the heme-bound NO is activated by the direct hydride transfer from NADH to generate a short-lived intermediate (<italic><underline>I</underline></italic>), a key state to promote N–N bond formation and N–O bond cleavage. This study applied time-resolved (TR) techniques in conjunction with photolabile-caged NO to gain direct experimental results for the characterization of the coordination and electronic structures of <italic><underline>I</underline></italic>. TR freeze-trap crystallography using an X-ray free electron laser (XFEL) reveals highly bent Fe–NO coordination in <italic><underline>I</underline></italic>, with an elongated Fe–NO bond length (Fe–NO = 1.91 Å, Fe–N–O = 138°) in the absence of NAD⁺. TR-infrared (IR) spectroscopy detects the formation of <italic><underline>I</underline></italic> with an N–O stretching frequency of 1,290 cm⁻¹ upon hydride transfer from NADH to the Fe³⁺–NO enzyme via the dissociation of NAD⁺ from a transient state, with an N–O stretching of 1,330 cm⁻¹ and a lifetime of ca. 16 ms. Quantum mechanics/molecular mechanics calculations, based on these crystallographic and IR spectroscopic results, demonstrate that the electronic structure of <italic><underline>I</underline></italic> is characterized by a singly protonated Fe³⁺–NHO^•− radical. The current findings provide conclusive evidence for the N₂O generation mechanism via a radical–radical coupling of the heme nitroxyl complex with the second NO molecule.

Mossbauer spectroscopy has been used to characterize oxygenated myoglobins (oxy Mbs) reconstituted with native and chemically modified Fe-57-enriched heme cofactors with different electron densities of the heme Fe atom (rho(Fe)) and to elucidate the effect of a change in the rho(Fe) on the nature of the bond between heme Fe and oxygen (O-2), i.e., the Fe-O-2 bond, in the protein. Quadrupole splitting (Delta E-Q) was found to decrease with decreasing rho(Fe), and the observed rho(Fe)-dependent Delta E-Q confirmed an increase in the contribution of the ferric-superoxide (Fe3+-O-2(-)) form to the resonance hybrid of the Fe-O-2 fragment with decreasing rho(Fe). These observations explicitly accounted for the lowering of O-2 affinity of the protein due to an increase in the O-2 dissociation rate and a decrease in the autoxidation reaction rate of oxy Mb through decreasing H+ affinity of the bound ligand with decreasing rho(Fe). Therefore, the present study demonstrated the mechanism underlying the electronic control of O-2 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 rho(Fe). Thus, the nature of the Fe-ligand bond in the protein was found to be affected by the rho(Fe).

<p>The reaction of Ni(<sc>ii</sc>)-(phenol)(phenolate) complexes with O₂ gave the Ni(<sc>ii</sc>)-phenoxyl radical complexes assisted by CH₃OH. This reaction was concluded to undergo <italic>via</italic> the proton transfer–electron transfer type mechanism without redox of the Ni ion.</p>

The dinuclear copper enzyme, tyrosinase, activates O2 to form a (μ-η2:η2-peroxido)dicopper(II) species, which hydroxylates phenols to catechols. However, the exact mechanism of phenolase reaction in the catalytic site of tyrosinase is still under debate. We herein report the near atomic resolution X-ray crystal structures of the active tyrosinases with substrate l-tyrosine. At their catalytic sites, CuA moved toward l-tyrosine (CuA1 → CuA2), whose phenol oxygen directly coordinates to CuA2, involving the movement of CuB (CuB1 → CuB2). The crystal structures and spectroscopic analyses of the dioxygen-bound tyrosinases demonstrated that the peroxide ligand rotated, spontaneously weakening its O−O bond. Thus, the copper migration induced by the substrate-binding is accompanied by rearrangement of the bound peroxide species so as to provide one of the peroxide oxygen atoms with access to the phenol substrate's ϵ carbon atom.

Metal complexes to promote oxidative DNA cleavage by H2O2 are desirable as anticancer drugs. A dicopper(II) complex of known p-cresol-derived methylene-tether ligand Hbcc [Cu2(bcc)]3+ did not promote DNA cleavage by H2O2. Here, we synthesized a new p-cresol-derived amide-tether one, 2,6-bis(1,4,7,10-tetrazacyclododecyl-1-carboxyamide)-p-cresol (Hbcamide). A dicopper(II) complex of the new ligand [Cu2(μ-OH)(bcamide)]2+ was structurally characterized. This complex promoted the oxidative cleavage of supercoiled plasmid pUC19 DNA (Form I) with H2O2 at pH 6.0-8.2 to give Forms II and III. The reaction was largely accelerated in a high pH region. A μ-1,1-hydroperoxo species was formed as the active species and spectroscopically identified. The amide-tether complex is more effective in cytotoxicity against HeLa cells than the methylene-tether one.

Large assemblies of respiratory chain complexes, known as supercomplexes, are present in the mitochondrial membrane in mammals and yeast, as well as in some bacterial membranes. The formation of supercomplexes is thought to contribute to efficient electron transfer, stabilization of each enzyme complex, and inhibition of reactive oxygen species (ROS) generation. In this study, mitochondria from various organisms were solubilized with digitonin, and then the solubilized complexes were separated by blue native PAGE (BN-PAGE). The results revealed a supercomplex consisting of complexes I, III, and IV in mitochondria from bovine and porcine heart, and a supercomplex consisting primarily of complexes I and III in mitochondria from mouse heart and liver. However, supercomplexes were barely detectable in Drosophila flight-muscle mitochondria, and only dimeric complex V was present. Drosophila mitochondria exhibited the highest rates of oxygen consumption and NADH oxidation, and the concentrations of the electron carriers, cytochrome c and quinone were higher than in other species. Respiratory chain complexes were tightly packed in the mitochondrial membrane containing abundant phosphatidylethanolamine with the fatty acid palmitoleic acid (C16:1), which is relatively high oxidation-resistant as compared to poly-unsaturated fatty acid. These properties presumably allow efficient electron transfer in Drosophila. These findings reveal the existence of a new mechanism of biological adaptation independent of supercomplex formation.

The design of protein oligomers with multiple active sites has been gaining interest, owing to their potential use for biomaterials, which has encouraged researchers to develop a new design method. Three-dimensional domain swapping is the unique phenomenon in which protein molecules exchange the same structural region between each other. Herein, to construct oligomeric heme proteins with different active sites by utilizing domain swapping, two c-type cytochrome-based chimeric proteins have been constructed and the domains swapped. According to X-ray crystallographic analysis, the two chimeric proteins formed a domain-swapped dimer with two His/Met coordinated hemes. By mutating the heme coordination structure of one of the two chimeric proteins, a domainswapped heterodimer with His/Met and His/H2O coordinated hemes was formed. Binding of an oxygen molecule to the His/H2O site of the heterodimer was confirmed by resonance Raman spectroscopy, in which the Fe-O-2 stretching band was observed at 580cm(-1) for the reduced/oxygenated heterodimer (at 554cm(-1) under an O-18(2) atmosphere). These results show that domain swapping is a useful method to design multiheme proteins.

Bovine cytochrome c oxidase (CcO), a 420-kDa membrane protein, pumps protons using electrostatic repulsion between protons transferred through a water channel and net positive charges created by oxidation of heme a (Fe-a) for reduction of O-2 at heme a(3) (Fe-a3). For this process to function properly, timing is essential: The channel must be closed after collection of the protons to be pumped and before Fea oxidation. If the channel were to remain open, spontaneous backflow of the collected protons would occur. For elucidation of the channel closure mechanism, the opening of the channel, which occurs upon release of CO from CcO, is investigated by newly developed time-resolved x-ray free-electron laser and infrared techniques with nanosecond time resolution. The opening process indicates that Cu-B senses completion of proton collection and binds O-2 before binding to Fe-a3 to close the water channel using a conformational relay system, which includes Cu-B, heme a(3), and a transmembrane helix, to block backflow of the collected protons.

An improved stopped-flow resonance Raman spectroscopy device was constructed using a stopped-flow mixer with a dead time of 3ms and a mixing volume of 0.1mL. The device was tested using myoglobin, where the formation reaction of a high-valent heme species, ferryl-oxo heme, was monitored by time-resolved resonance Raman spectroscopy after mixing a ferric myoglobin solution with a hydrogen peroxide solution. The ferryl-oxo heme formation rate constant obtained by Raman spectroscopy is in good agreement with the rate constant obtained by conventional stopped-flow absorption spectroscopy for the same reaction under the same conditions. It is proved by these results that the present device is generally applicable to enzyme-substrate reactions with a significantly higher time resolution than previously reported. Copyright (c) 2017 John Wiley & Sons, Ltd.

To understand the roles of mitochondrial respiratory chain supercomplexes, methods for consistently separating and preparing supercomplexes must be established. To this end, we solubilized supercomplexes from bovine heart mitochondria with digitonin and then replaced digitonin with amphipol (A8-35), an amphiphilic polymer. Afterward, supercomplexes were separated from other complexes by sucrose density gradient centrifugation. Twenty-six grams of bovine myocardium yielded 3.2 mg of amphipol-stabilized supercomplex. The purified supercomplexes were analyzed based on their absorption spectra as well as Q(10) (ubiquinone with ten isoprene units) and lipid assays. The supercomplex sample did not contain cytochrome c but did contain complexes I, III, and IV at a ratio of 1:2:1, 6 molecules of Q(10), and 623 atoms of phosphorus. When cytochrome c was added, the supercomplex exhibited KCN-sensitive NADH oxidation; thus, the purified supercomplex was active. Reduced complex IV absorbs at 444 nm, so we measured the resonance Raman spectrum of the reduced amphipol-solubilized supercomplex and the mixture of amphipol-solubilized complexes I-1, III2, and IV1 using an excitation wavelength of 441.6 nm, allowing measurement precision comparable with that obtained for complex IV alone. Use of the purified active sample provides insights into the effects of supercomplex formation.

We analyzed the oxygen (O-2) 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 (rho(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, S3, 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 O-2 affinity and autoxidation reaction rate of the H64Q mutant decreased with a decrease in rho(Fe) as expected from the effect of a change in rho(Fe) on the resonance between the Fe2+-O-2 bond and Fe3+-O-2-like species in the O-2 form, while the CO affinity of the protein is independent of a change in rho(Fe). We also found that the well-known inverse correlation between the frequencies of Fe-bound CO (nu(CO)) and Fe-C (nu(FeC)) stretching [Li, X.-Y.; Spiro, T. G. J. Am. Chem. Soc. 1988, 110, 6024-6033] is affected differently by changes in rho(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.

UV-visible absorption spectroscopy is useful for probing the electronic and structural changes of protein active sites, and thus the on-line combination of X-ray diffraction and spectroscopic analysis is increasingly being applied. Herein, a novel absorption spectrometer was developed at SPring-8 BL26B2 with a nearly on-axis geometry between the X-ray and optical axes. A small prism mirror was placed near the X-ray beamstop to pass the light only 2 degrees off the X-ray beam, enabling spectroscopic analysis of the X-ray-exposed volume of a crystal during X-ray diffraction data collection. The spectrometer was applied to NO reductase, a heme enzyme that catalyzes NO reduction to N2O. Radiation damage to the heme was monitored in real time during X-ray irradiation by evaluating the absorption spectral changes. Moreover, NO binding to the heme was probed via caged NO photolysis with UV light, demonstrating the extended capability of the spectrometer for intermediate analysis.

A set of nickel(III) peroxo complexes bearing tetraazamacrocyclic ligands, [Ni-III(TBDAP)(O-2)](+) (TBDAP = N,Ni-di-tert-butyl-2,11-diaza[3.3](2,6)pyridinophane) and [Ni-III(CHDAP)(O-2)](+) (CHDAP = N,N'-dicyclohexyl-2,11-diaza[3.3](2,6)pyridinophane), were prepared by reacting [Ni-II(TBDAP)(NO3)(H2O)](+) and [Ni-II(CHDAP)(NO3)](+), respectively, with H2O2 in the presence of triethylamine. The mononuclear nickel(III) peroxo complexes were fully chatacterized by various physicochemical methods, such as UV-vis, electrospray ionization mass spectrometry, resonance Raman, electron paramagnetic resonance, and X-ray analysis. The spectroscopic and structural characterization clearly shows that the NiO2 cores are almost identical where the peroxo ligand is bound in a side-on fashion. properties of the supporting ligands were confirmed by X-ray crystallography, where the CHDAP ligand gives enough space around the Ni core compared to the TBDAP ligand. The nickel(III) peroxo complexes showed reactivity in the oxidation of aldehydes. In the aldehyde deformylation reaction, the nucleophilic reactivity of the nickel(III) peroxo, complexes was highly dependent on the steric properties of the macrocyclic ligands, with. a reactivity order of [Ni-III(TBDAP)(O-2)](+) &lt; [Ni-III(CHDAP)(O-2)](+). This result provides fundamental insight into the mechanism of the structure (steric) reactivity relationship of metal peroxo intermediates.

A conventional method for reconstituting cytochrome c oxidase (CcO) into phospholipid vesicles (COAT) has been modified to permit resonance Raman (RR) analysis in the presence and absence of proton motive force (Delta mu(+)(H)). The COV has an average diameter of 20 nm and contains one CcO molecule within a unified orientation with Cu-A located outside the COV. The process of generation of Delta mu(+)(H) across the membrane was monitored spectrophotometrically with rhodamine123 dye. The COV exhibits a respiratory control ratio (RCR) value of &gt;30 and is tolerant to RR measurements with 10 mW laser illumination for 60 min at 441.6 nm. Structural perturbations at the heme sites caused by incorporation into vesicles were clarified by spectral comparisons between solubilized CcO and COV. Absorption spectroscopy revealed that the rate of electron transfer from cytochrome c to O-2 is reduced significantly more in the presence of Delta mu(+)(H) than in its absence. RR spectroscopic measurements indicate that CcO in COV in the "respiratory-controlled" state adopts a mixed-valence state (heme a(2+) and heme a(3)(3+)). This study establishes a supramolecular model system for experimentally examining the energy conversion protein machinery in the presence of Delta mu(+)(H).

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 (O-2), possibly due to stabilization of the heme Fe atom-bound O-2 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 O-2 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 (rho(Fe)) on the O-2 versus CO discrimination. The study demonstrated that the preferential binding of O-2 over CO by the protein was achieved through increasing rho(Fe), and the ordinary ligand-binding preference, that is, the preferential binding of CO over O-2, by the protein was achieved through decreasing rho(Fe). Thus, the O-2 and CO binding preferences of the L29F mutant protein could be controlled through electronic modulation of intrinsic heme Fe reactivity through a change in rho(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.

The stretching frequency of the coordination bond between the heme Fe atom and proximal histidine (His93) N-epsilon atom (nu(Fe-His)), and the NMR shift of the His93N(delta)H proton (His93N(delta)H shift) of the deoxy form of a hemoprotein have been used to determine the electronic nature of the His93 imidazole highly relevant to regulation of heme Fe reactivity. We determined the nu(Fe-His) values and His93N(delta)H shifts of the deoxy forms of myoglobins reconstituted with artificial heme cofactors possessing strongly electron-withdrawing trifluoromethyl (CF3) group(s) as peripheral side chain(s). The study revealed that the bond between the heme Fe and His93 becomes stronger with increasing number of CF3 substitutions due to an increase in acidity of the His93N(delta)H hydrogen bonded to the carbonyl O atom of Leu89. Thus, the present study demonstrated that the electronic nature of the His93 imidazole in deoxy Mb is affected by electronic perturbation induced by chemical modification of the heme cofactor.

Resonance Raman spectra of ligand-bound complexes including the 4-phenylimidazole complex and of free and L-Trp-bound forms of indoleamine 2, 3-dioxygenase in the ferric state were examined. Effects on the vinyl and propionate substituent groups of the heme were detected in a ligand-dependent fashion. The effects of phenyl group of 4-phenylimidazole on the vinyl and propionate Raman bands were evident when compared with the case of imidazole ligand. Substrate binding to the ferrous protein caused an upshift of the iron-histidine stretching mode by 3 cm(-1), indicating an increase in negativity of the imidazole ring, which favors the O-O bond cleavage. The substrate binding event is likely to be communicated from the heme distal side to the iron-histidine bond through heme substituent groups and the hydrogen-bond network which includes water molecules, as identified in an X-ray structure of a 4-phenylimidazole complex. The results provide evidence for fine-tuning of the reactivity of O-O bond cleavage by the oxygenated heme upon binding of L-Trp. (C) 2013 Elsevier B. V. All rights reserved.

We have previously shown that methionine-heme iron coordination is perturbed in domain-swapped dimeric horse cytochrome c. To gain insight into the effect of methionine dissociation in dimeric cytochrome c, we investigated its interaction with cyanide ion. We found that the Soret and Q bands of oxidized dimeric cytochrome c at 406.5 and 529 nm redshift to 413 and 536 nm, respectively, on addition of 1 mM cyanide ion. The binding constant of dimeric cytochrome c and cyanide ion was obtained as 2.5 x 10(4) M-1. The Fe-CN and C-N stretching (nu (Fe-CN) and nu (CN)) resonance Raman bands of CN--bound dimeric cytochrome c were observed at 443 and 2,126 cm(-1), respectively. The nu (Fe-CN) frequency of dimeric cytochrome c was relatively low compared with that of other CN--bound heme proteins, and a relatively strong coupling between the Fe-C-N bending and porphyrin vibrations was observed in the 350-450-cm(-1) region. The low nu (Fe-CN) frequency suggests weaker binding of the cyanide ion to dimeric cytochrome c compared with other heme proteins possessing a distal heme cavity. Although the secondary structure of dimeric cytochrome c did not change on addition of cyanide ion according to circular dichroism measurements, the dimer dissociation rate at 45 A degrees C increased from (8.9 +/- A 0.7) x 10(-6) to (3.8 +/- A 0.2) x 10(-5) s(-1), with a decrease of about 2 A degrees C in its dissociation temperature obtained with differential scanning calorimetry. The results show that diatomic ligands may bind to the heme iron of dimeric cytochrome c and affect its stability.

A hypochloritoiron(III) porphyrin species has been proposed as a key intermediate in an antimicrobial defense system in neutrophils and in hemecatalyzed chlorination reactions. We report herein the preparation, spectroscopic characterization, and reactivity of the bis(hypochlorito)iron(III) porphyrin complex [(TPFP)Fe-III(OC1)(2)](-) (1) and the imidazole-hypochloritoiron complexes (TPFP)Fe-III(OC1)(1-R-Im) [R = CH3 (2), H (3), CH2CO2H (4)], in which TPFP is 5,10,15,20-tetrakis(pentafluorophenyl)porphyrinate. The structures of 1-4 were confirmed by absorption, H-2 and F-19 NMR, EPR, and resonance Raman spectroscopy and electrospray ionization mass spectrometry at low temperature. The reactions of 1 and 2 with various organic substrates show that 1 and 2 are capable of chlorination, sulfoxidation, and epoxidation reactions and that 1 is much more reactive with these substrates than 2.

The hydrophobic patch of, azurin (AZ) from Pseudomonas aeruginosa is an important recognition surface for electron transfer (ET) reactions. The influence of changing the size of this region, by mutating the C-terminal copper binding loop, on the ET reactivity of AZ adsorbed on gold electrodes modified with alkanethiol self-assembled monolayers (SAMs) has been studied. The distance-dependence of ET kinetics measured by cyclic voltammetry using SAMs of variable chain length, demonstrates that the activation barrier for short-range ET is dominated by the dynamics of molecular rearrangements accompanying ET at the AZ-SAM interface. These include internal electric field dependent low amplitude protein motions and the reorganization of interfacial water molecules, but not protein reorientation. Interfacial molecular dynamics also control the kinetics of short-range ET for electrostatically and covalently immobilized cytochrome c. This mechanism therefore may be utilized for short-distance ET irrespective of the type of metal center, the surface electrostatic potential, and the nature of the protein- SAM interaction.

The copper site and overall structures of azurin (AZ) variants in which the amicyanin (AMI) and plastocyanin (PC) metal binding loops have been introduced, AZAMI and AZPC, respectively, are similar to that of AZ, whereas the loop conformations resemble those in the native proteins. To assess the influence of these loop mutations on stability, the thermal unfolding of AZAMI and AZPC has been investigated by differential scanning calorimetry, absorption and fluorescence spectroscopy. The calorimetric profiles of both variants exhibit a complex shape consisting of two endothermic peaks and an exothermic peak. The temperature of the maximum heat of absorption for the single endothermic peak is 82.7 degrees C for AZ, whereas for AZAMI and AZPC the most intense endothermic peaks are at 74.9 and 68.1 degrees C comparable to values for AMI and PC, respectively. Denaturation investigated using the temperature dependence of the absorbance at similar to 600 nm and Trp emission, also demonstrates decreased stability for both loop mutants. The thermal transition between the native and the denaturated states is irreversible, scan rate dependent and consistent with the two-state irreversible model. The structure of the active-site loop has a dramatic effect on the kinetic stability and the unfolding pathway of cupredoxins. (C) 2012 Elsevier Inc. All rights reserved.

Autocatalytic formation of His-Cys cross-linkage in the enzyme active site of tyrosinase from Aspergillus oryzae has been demonstrated to proceed by the treatment of apoenzyme with Cu(II) under aerobic conditions, where a (mu-eta(2):eta(2)-peroxo)dicopper(II) species has been suggested to be involved as a key reactive intermediate.

Resonance Raman (RR) spectra of the oxygenated and Fe-IV=O reaction intermediates of indoleamine 2,3-dioxygenase (IDO) are reported. Absorption and RR spectra reveal that the electronic and geometric structures of the two respective species at pH 6.5 and pH 8.0 are the same, although the enzymatic activity at pH 6.5 is 6 times higher than at pH 8.0. The results thus further support our current understanding that the Fe-IV=O heme species is the active species in the IDO reaction cycle, although its presence was unexpected. The Fe-O-2 and the O-O stretching frequencies of the IDO-Trp-O-2 ternary complex at Trp concentrations of 50 mu M and 8 mM are essentially identical. These results suggest that "substrate inhibition'' of enzymatic activity occurs by binding of a second substrate molecule to an unknown binding site and not to the heme pocket.

Indoleamine 2,3-dioxygenase (IDO) is a heme enzyme which catalyzes dioxygenation of L-Trp (tryptophan), yielding N-formylkynurenine. IDO thus plays a key role in L-Trp catabolism in mammals. In the present study, resonance Raman (RR) spectra of the reduced carbon monoxide- (CO-) bound form of IDO were measured in order to gain insights into the active site environment of O(2). Binding of CO to L-Trp-bound IDO causes a significant change in the electronic and RR spectra of the heme, indicating that the pi* orbitals of the carbon atom of CO interact with pi orbitals of Fe and the porphyrin. On the other hand, binding of CO to D-Trp-bound IDO does not induce the same change. This is also the case with substrate-free IDO. Based on the distinct absorption spectra and RR bands of the vibrational signature of CO (v(CO), delta(FeCO), and v(Fe-CO)) of the L-Trp-bound species relative to the other two species, it is confirmed that sterically constrained geometry of the Fe-O-O unit exists as previously reported (Terentis, A. C., et al. (2002) J. Biol. Chem. 277, 15788-15794). In contrast, binding of D-Trp does not induce such constraint. The comparable values of V(max) reported for L-Trp and D-Trp are interpreted as a result of a change in the rate-limiting step in the reaction cycle of the enzyme induced by the D-enantiomer relative to the L-enantiomer. Enhancements of the overtone and the combination Raman modes of the Fe-CO stretching vibration are evident. The anharmonicity of the Fe-CO stretching oscillator is significantly higher than those of oxygen carrier proteins. This is a specific character of IDO and might be responsible for the unique reactivity of this enzyme.