久保 稔

Abstract Photosystem II (PSII) catalyses the oxidation of water through a four-step cycle of S_i states (i = 0–4) at the Mn₄CaO₅ cluster^1–3, during which an extra oxygen (O6) is incorporated at the S₃ state to form a possible dioxygen^4–7. Structural changes of the metal cluster and its environment during the S-state transitions have been studied on the microsecond timescale. Here we use pump-probe serial femtosecond crystallography to reveal the structural dynamics of PSII from nanoseconds to milliseconds after illumination with one flash (1F) or two flashes (2F). Y_Z, a tyrosine residue that connects the reaction centre P680 and the Mn₄CaO₅ cluster, showed structural changes on a nanosecond timescale, as did its surrounding amino acid residues and water molecules, reflecting the fast transfer of electrons and protons after flash illumination. Notably, one water molecule emerged in the vicinity of Glu189 of the D1 subunit of PSII (D1-E189), and was bound to the Ca²⁺ ion on a sub-microsecond timescale after 2F illumination. This water molecule disappeared later with the concomitant increase of O6, suggesting that it is the origin of O6. We also observed concerted movements of water molecules in the O1, O4 and Cl-1 channels and their surrounding amino acid residues to complete the sequence of electron transfer, proton release and substrate water delivery. These results provide crucial insights into the structural dynamics of PSII during S-state transitions as well as O–O bond formation.

Abstract Understanding and controlling protein motion at atomic resolution is a hallmark challenge for structural biologists and protein engineers because conformational dynamics are essential for complex functions such as enzyme catalysis and allosteric regulation. Time-resolved crystallography offers a window into protein motions, yet without a universal perturbation to initiate conformational changes the method has been limited in scope. Here we couple a solvent-based temperature jump with time-resolved crystallography to visualize structural motions in lysozyme, a dynamic enzyme. We observed widespread atomic vibrations on the nanosecond timescale, which evolve on the submillisecond timescale into localized structural fluctuations that are coupled to the active site. An orthogonal perturbation to the enzyme, inhibitor binding, altered these dynamics by blocking key motions that allow energy to dissipate from vibrations into functional movements linked to the catalytic cycle. Because temperature jump is a universal method for perturbing molecular motion, the method demonstrated here is broadly applicable for studying protein dynamics.

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.

Abstract 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.

Significance Light-driven chloride pumps have been identified in various species, including archaea and marine flavobacteria. The function of ion transportation controllable by light is utilized for optogenetics tools in neuroscience. Chloride pumps differ among species, in terms of amino acid homology and structural similarity. Our time-resolved crystallographic studies using X-ray free electron lasers reveal the molecular mechanism of halide ion transfer in a light-driven chloride pump from a marine flavobacterium. Our data indicate a common mechanism in chloride pumping rhodopsins, as compared to previous low-temperature trapping studies of chloride pumps. These findings are significant not only for further improvements of optogenetic tools but also for a general understanding of the ion pumping mechanisms of microbial rhodopsins.

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.

Photosystem II (PSII) catalyzes light-induced water oxidation through an S<italic> _i </italic>-state cycle, leading to the generation of di-oxygen, protons and electrons. Pump–probe time-resolved serial femtosecond crystallography (TR-SFX) has been used to capture structural dynamics of light-sensitive proteins. In this approach, it is crucial to avoid light contamination in the samples when analyzing a particular reaction intermediate. Here, a method for determining a condition that avoids light contamination of the PSII microcrystals while minimizing sample consumption in TR-SFX is described. By swapping the pump and probe pulses with a very short delay between them, the structural changes that occur during the S₁-to-S₂ transition were examined and a boundary of the excitation region was accurately determined. With the sample flow rate and concomitant illumination conditions determined, the S₂-state structure of PSII could be analyzed at room temperature, revealing the structural changes that occur during the S₁-to-S₂ transition at ambient temperature. Though the structure of the manganese cluster was similar to previous studies, the behaviors of the water molecules in the two channels (O1 and O4 channels) were found to be different. By comparing with the previous studies performed at low temperature or with a different delay time, the possible channels for water inlet and structural changes important for the water-splitting reaction were revealed.

Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free electron laser, which revealed conformational changes following photoactivation. The isomerized retinal adopts a twisted conformation and shifts toward the putative internal proton donor residues, consequently inducing an outward shift of TM3, as well as a local deformation in TM7. These early conformational changes in the pore-forming helices should be the triggers that lead to opening of the ion conducting pore.

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme.

Inspecting S states in photosynthesis Oxygenic photosynthesis uses a Mn ₄ CaO ₅ cluster in the oxygen-evolving complex to extract electrons from water and produce dioxygen. Visualizing each of the chemical states in this process, S ₀ to S ₄ , and assigning chemical identities and mechanisms on the basis of structures has been a challenge addressed recently by work at x-ray free-electron lasers. Suga et al. used serial crystallography at cryogenic temperatures to trap and determine the structures of several stable states during photosystem II water oxidation (see the Perspective by Britt and Marchiori). Changes around the water cluster already happen in the S ₂ state and set the stage for water insertion that occurs during transition to the S ₃ state. A short 1.9-angstrom distance between the two oxygen atoms in the S ₃ state is consistent with theoretical studies supporting an oxyl/oxo mechanism for oxygen-oxygen coupling. Science , this issue p. 334 ; see also p. 305

<p>Reorganization energies (λ) of electron transfer (ET) and proton-coupled ET (PCET) from electron donors to isolated Ru^IV(O) complexes were determined to be in the range of 1.70–1.88 eV (ET) and 1.20–1.26 eV (PCET).</p>

Single-crystal infrared (IR) spectroscopy is a promising method for protein structure analysis, where a protein crystal sample is fixed in a micro flow cell. Single-crystal calcium fluoride (CaF2) is expected as the flow cell substrate material for its excellent optical property. However, CaF2 is a highly brittle material having strong anisotropy, thus is extremely difficult to machine. Up to date, there is no available literature on fabrication of CaF2 flow cells. In this study, micro flow cells of single-crystal CaF2 were fabricated by ultraprecision cutting technology. Fly cutting was conducted using a single-crystal diamond tool having straight edges to generate a depth-varying rectangle cross section for the flow cell. The effects of cutting direction, workpiece orientation, undeformed chip thickness and tool rake angle on cutting behavior were investigated. Based on experiments and analysis, optimal conditions for ductile machining of micro grooves in CaF2 were identified. As a result, a 10 μm deep CaF2 micro flow cell with surface roughness of 2.4 nmRa was successfully fabricated. Using the fabricated flow cell, IR spectroscopic analysis of a protein single crystal at room temperature was succeeded. This study demonstrated the effectiveness of ultraprecision cutting technology in CaF2 micro flow cell fabrication, which contributes to the IR analysis of protein, and in turn, the advance of life science.

Time-resolved serial femtosecond crystallography using an X-ray free electron laser (XFEL) in conjunction with a photosensitive caged-compound offers a crystallographic method to track enzymatic reactions. Here we demonstrate the application of this method using fungal NO reductase, a heme-containing enzyme, at room temperature. Twenty milliseconds after caged-NO photolysis, we identify a NO-bound form of the enzyme, which is an initial intermediate with a slightly bent Fe-N-O coordination geometry at a resolution of 2.1 angstrom. The NO geometry is compatible with those analyzed by XFEL-based cryo-crystallography and QM/MM calculations, indicating that we obtain an intact Fe3+-NO coordination structure that is free of X-ray radiation damage. The slightly bent NO geometry is appropriate to prevent immediate NO dissociation and thus accept H- from NADH. The combination of using XFEL and a caged-compound is a powerful tool for determining functional enzyme structures during catalytic reactions at the atomic level.

X-ray free-electron lasers (XFELs) have opened new opportunities for timeresolved X-ray crystallography. Here a nanosecond optical-pump XFEL-probe device developed for time-resolved serial femtosecond crystallography (TRSFX) studies of photo-induced reactions in proteins at the SPring-8 Angstrom Compact free-electron LAser (SACLA) is reported. The optical-fiber-based system is a good choice for a quick setup in a limited beam time and allows pump illumination from two directions to achieve high excitation efficiency of protein microcrystals. Two types of injectors are used: one for extruding highly viscous samples such as lipidic cubic phase (LCP) and the other for pulsed liquid droplets. Under standard sample flow conditions from the viscous-sample injector, delay times from nanoseconds to tens of milliseconds are accessible, typical time scales required to study large protein conformational changes. A first demonstration of a TR-SFX experiment on bacteriorhodopsin in bicelle using a setup with a droplet-type injector is also presented.

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.

Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer. It catalyses light-driven water oxidation at its catalytic centre, the oxygen-evolving complex (OEC)(1-3). The structure of PSII has been analysed at 1.9 angstrom resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 cluster organized in an asymmetric, `distorted-chair' form(4). This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the `radiation damage-free'(5) structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temperature at a resolution of 2.35 angstrom using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 angstrom compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the QB/non-haem iron and the Mn4CaO5 cluster. The changes around the QB/non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water molecule located 3.5 angstrom from the Mn4CaO5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water molecule and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique mu 4-oxo-bridge located in the quasi-centre of Mn1 and Mn4 (refs 4,5). This suggests the insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 angstrom between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previously(6,7.)

Bacteriorhodopsin (bR) is a light-driven proton pump and a model membrane transport protein. We used time-resolved serial femtosecond crystallography at an x-ray free electron laser to visualize conformational changes in bR from nanoseconds to milliseconds following photoactivation. An initially twisted retinal chromophore displaces a conserved tryptophan residue of transmembrane helix F on the cytoplasmic side of the protein while dislodging a key water molecule on the extracellular side. The resulting cascade of structural changes throughout the protein shows how motions are choreographed as bR transports protons uphill against a transmembrane concentration gradient.

CooA is a CO-sensing transcriptional activator from the photosynthetic bacterium Rhodospirillum rubrum that binds CO at the heme iron. The heme iron in ferrous CooA has two axial ligands: His77 and Pro2. CO displaces Pro2 and induces a conformational change in CooA. The dissociation of CO and/or ligation of the Pro2 residue are believed to trigger structural changes in the protein. Visible time-resolved resonance Raman spectra obtained in this study indicated that the nu(Fe-His) mode, arising from the proximal His77 iron stretch, does not shift until 50 after the photodissociation of CO. Ligation of the Pro2 residue to the heme iron was observed around 50 its after the photodissociation of CO, suggesting that the nu(Fe-His) band exhibits no shift until the ligation of Pro2. UV resonance Raman spectra suggested structural changes in the vicinity of Trp110 in the C-helix upon binding, but no or very small spectral changes in the time-resolved UV resonance Raman spectra were observed from 100 ns to 100 its after the photodissociation of CO. These results strongly suggest that the conformational change of CooA is induced by the ligation of Pro2 to the heme iron.

Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 angstrom resolution and a method to investigate protein dynamics with modest sample requirement. Time-resolved SFX (TR-SFX) with a pump-probe delay of 1ms yields difference Fourier maps compatible with the dark to M state transition of bR. Importantly, the method is very sample efficient and reduces sample consumption to about 1mg per collected time point. Accumulation of M intermediate within the crystal lattice is confirmed by time-resolved visible absorption spectroscopy. This study provides an important step towards characterizing the complete photocycle dynamics of retinal proteins and demonstrates the feasibility of a sample efficient viscous medium jet for TR-SFX.

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.

Background: Cytochrome c oxidase reduces O-2 coupled with proton pumping. Results: A newly developed time-resolved infrared system reveals transient conformational changes in the proton-pumping pathway upon CO binding to Cu-B in the O-2 reduction site. Conclusion: Cu-B promotes proton collection and effective blockage of back-leak of pumping protons. Significance: These critical findings in bioenergetics stimulate the new infrared approach for mechanistic investigation of any other protein function. X-ray structural and mutational analyses have shown that bovine heart cytochrome c oxidase (CcO) pumps protons electrostatically through a hydrogen bond network using net positive charges created upon oxidation of a heme iron (located near the hydrogen bond network) for O-2 reduction. Pumping protons are transferred by mobile water molecules from the negative side of the mitochondrial inner membrane through a water channel into the hydrogen bond network. For blockage of spontaneous proton back-leak, the water channel is closed upon O-2 binding to the second heme (heme a(3)) after complete collection of the pumping protons in the hydrogen bond network. For elucidation of the structural bases for the mechanism of the proton collection and timely closure of the water channel, conformational dynamics after photolysis of CO (an O-2 analog)-bound CcO was examined using a newly developed time-resolved infrared system feasible for accurate detection of a single C=O stretch band of -helices of CcO in H2O medium. The present results indicate that migration of CO from heme a(3) to Cu-B in the O-2 reduction site induces an intermediate state in which a bulge conformation at Ser-382 in a transmembrane helix is eliminated to open the water channel. The structural changes suggest that, using a conformational relay system, including Cu-B, O-2, heme a(3), and two helix turns extending to Ser-382, Cu-B induces the conformational changes of the water channel that stimulate the proton collection, and senses complete proton loading into the hydrogen bond network to trigger the timely channel closure by O-2 transfer from Cu-B to heme a(3).

Redox properties of a mononuclear copper(II) superoxide complex, (L)Cu II-OO•, supported by a tridentate ligand (L = 1-(2-phenethyl)-5-[2-(2-pyridyl)ethyl]-1,5-diazacyclooctane) have been examined as a model compound of the putative reactive intermediate of peptidylglycine α-hydroxylating monooxygenase (PHM) and dopamine β-monooxygenase (DβM) (Kunishita et al. J. Am. Chem. Soc. 2009, 131, 2788-2789 Inorg. Chem. 2012, 51, 9465-9480). On the basis of the reactivity toward a series of one-electron reductants, the reduction potential of (L)CuII-OO • was estimated to be 0.19 ± 0.07 V vs SCE in acetone at 298 K (cf. Tahsini et al. Chem. - Eur. J. 2012, 18, 1084-1093). In the reaction of TEMPO-H (2,2,6,6-tetramethylpiperidine-N-hydroxide), a simple HAT (hydrogen atom transfer) reaction took place to give the corresponding hydroperoxide complex LCuII-OOH, whereas the reaction with phenol derivatives (XArOH) gave the corresponding phenolate adducts, LCu II-OXAr, presumably via an acid-base reaction between the superoxide ligand and the phenols. The reaction of (L)CuII-OO • with a series of triphenylphosphine derivatives gave the corresponding triphenylphosphine oxides via an electrophilic ionic substitution mechanism with a Hammett ρ value as -4.3, whereas the reaction with thioanisole (sulfide) only gave a copper(I) complex. These reactivities of (L)CuII-OO• are different from those of a similar end-on superoxide copper(II) complex supported by a tetradentate TMG 3tren ligand (1,1,1-Tris{2-[N2-(1,1,3,3- tetramethylguanidino)]ethyl}amine (Maiti et al. Angew. Chem., Int. Ed. 2008, 47, 82-85). © 2013 American Chemical Society.

The carbomethoxy substituted dithiolene ligand (L-COOMe) enabled us to develop a series of new bis(ene-1,2- dithiolato) tungsten complexes including (WO)-O-VI, W-IV(OSiBuPh2), (WO2)-O-VI, (WO)-O-VI(OSiBuPh2) and (WO)-O-VI(S) core structures. By using these tungsten complexes, a systematic study of the terminal monodentate ligand effects has been performed on the structural, spectroscopic properties and reactivity. The structure and spectroscopic properties of the tungsten complexes have also been compared to those of the molybdenum complexes coordinated by the same ligand to investigate the effects of the metal ion (W vs. Mo). X-ray crystallographic analyses of the tungsten(IV) complexes have revealed that the tungsten centres adopt a distorted square pyramidal geometry with a dithiolene ligand having an ene-1,2-dithiolate form. On the other hand, the dioxotungsten(VI) complex exhibits an octahedral structure consisting of the bidentate L-COOMe and two oxo groups, in which p-delocalization was observed between the (WO2)-O-VI and ene-1,2-dithiolate units. The tungsten(IV) and dioxotungsten(VI) complexes are isostructural with the molybdenum counter parts. DFT calculation study of the (WO)-O-VI(S) complex has indicated that the W=S bond of 2.2 angstrom is close to the bond length between the tungsten centre and ambiguously assigned terminal monodentate atom in aldehyde oxidoreductase of the tungsten enzyme. Resonance Raman (rR) spectrum of the (WO)-O-VI(S) complex has shown the two inequivalent L-COOMe ligands with respect to their bonding interactions with the tungsten centre, reproducing the appearance of two upsilon(C=C) stretches in the rR spectrum of aldehyde oxidoreductase. Sulfur atom transfer reaction from the (WO)-O-VI(S) complex to triphenylphosphines has also been studied kinetically to demonstrate that the tungsten complex has a lower reactivity by about one-order of magnitude, when compared with its molybdenum counterpart.

Oxo-sulfido- and oxo-selenido-molybdenum(VI) complexes with an ene-1,2-dithiolate ligand are generated as models of the active sites of molybdenum hydroxylases. The sulfide and selenide groups are highly reactive toward triphenylphosphine in the order of Se &gt; S.

Reaction of a copper(I) complex supported by a sterically demanding tripodal tetradentate ligand, HIPT3tren, and O2 gave a mononuclear copper(II) end-on superoxo complex. Spectroscopic (UV/Vis, resonance Raman, ESR, and 1H-NMR) and DFT studies have been performed. The O2-binding process as well as the reaction toward external substrates have been investigated kinetically to demonstrate the unique behavior of the copper(II) end-on superoxo complex, which may occur as a result of the existence of the hydrophobic core around the copper coordination sphere created by the HIPT3tren ligand.

The O-2 and NO reactivity of a Cr(II) complex bearing a 12-membered tetraazamacrocyclic N-tetramethylated cyclam (TMC) ligand, [Cr-II(12-TMC)-(Cl)](+) (1), and the NO reactivity of its peroxo derivative, [Cr-IV(12-TMC)(O-2)(Cl)](+) (2), are described. By contrast to the previously reported Cr(III)-superoxo complex, [Cr-III(14-TMC)(O-2)(Cl)](+), the Cr(IV)-peroxo complex 2 is formed in the reaction of 1 and O-2. Full spectroscopic and X-ray analysis revealed that 2 possesses side-on eta(2)-peroxo ligation. The quantitative reaction of 2 with NO affords a reduction in Cr oxidation state, producing a Cr(III)-nitrato complex, [Cr-III(12-TMC)(NO3)(Cl)](+) (3). The latter is suggested to form via a Cr(III)- peroxynitrite intermediate. [Cr-II(12-TMC)(NO)(Cl)](+) (4), a Cr(II)-nitrosyl complex derived from 1 and NO, could also be synthesized; however, it does not react with O-2.

Normal vibrational modes of heme have been analyzed using density functional theory, to assign a new Raman band observed at 312 cm(-1) in the CO-bound, active form of soluble guanylate cyclase. The conserved YxSxR motif is incorporated into the model to reproduce the hydrogen-bonding network around propionates. A delocalized mode that involves Arg motions as well as pyrrole tilting and propionate bending is found to be the most likely candidate accounting for the new band.

HemAT-Bs is a heme-based signal transducer protein responsible for aerotaxis. Time-resolved ultraviolet resonance Raman (UVRR) studies of wild-type and Y70F mutant of the full-length HemAT-Bs and the truncated sensor domain were performed to determine the site-specific protein dynamics following carbon monoxide (CO) photodissociation. The UVRR spectra indicated two phases of intensity changes for Trp, Tyr, and Phe bands of both full-length and sensor domain proteins. The W16 and W3 Raman bands of Trp, the F8a band of Phe, and the Y8a band of Tyr increased in intensity at hundreds of nanoseconds after CO photodissociation, and this was followed by recovery in similar to 50 mu s. These changes were assigned to Trp-132 (G-helix), Tyr-70 (B-helix), and Phe-69 (B-helix) and/or Phe-137 (G-helix), suggesting that the change in the heme structure drives the displacement of B- and G-helices. The UVRR difference spectra of the sensor domain displayed a positive peak for amide I in hundreds of nanoseconds after photolysis, which was followed by recovery in similar to 50 mu s. This difference band was absent in the spectra of the full-length protein, suggesting that the isolated sensor domain undergoes conformational changes of the protein backbone upon CO photolysis and that the changes are restrained by the signaling domain. The time-resolved difference spectrum at 200 mu s exhibited a pattern similar to that of the static (reduced - CO) difference spectrum, although the peak intensities were much weaker. Thus, the rearrangements of the protein moiety toward the equilibrium ligand-free structure occur in a time range of hundreds of microseconds.

Protein dynamics on the subnanosecond to microsecond time scale was investigated for the isolated heme domain of a gas sensor protein, EcDOS, with time-resolved ultraviolet resonance Raman (UVRR) spectroscopy. Rapid structural changes (&lt;0.5 ns) due to CO dissociation and nanosecond structural relaxation following geminate recombination of CO were observed through a Raman band of Trp53 located near the heme. Microsecond transient UVRR spectra showed several phases of intensity changes in both Tip and Tyr bands. In hundreds of nanoseconds after CO photodissociation, the W18, W16, and W3 bands of Trp residues and Y8a band of Tyr residues decreased in intensity and were followed by the intensity recovery of Tyr band in 50 mu s and of Tip bands in hundreds microseconds. This observation demonstrates that a change in the heme ligation triggers conformational changes in the protein moiety through heme side chains.

Nuclear resonance vibrational spectroscopy (NRVS) reveals the vibrational dynamics of a Mossbauer probe nucleus. Here, Fe-57 NRVS measurements yield the complete spectrum of Fe vibrations in halide complexes of iron porphyrins. Iron porphine serves as a useful symmetric model for the more complex spectrum of asymmetric heme molecules that contribute to numerous essential biological processes. Quantitative comparison with the vibrational density of states (VDOS) predicted for the Fe atom by density functional theory calculations unambiguously identifies the correct sextet ground state in each case. These experimentally authenticated calculations then provide detailed normal mode descriptions for each observed vibration. All Fe-ligand vibrations are clearly identified despite the high symmetry of the Fe environment. Low frequency molecular distortions and acoustic lattice modes also contribute to the experimental signal. Correlation matrices compare vibrations between different molecules and yield a detailed picture of how heme vibrations evolve in response to (a) halide binding and (b) asymmetric placement of porphyrin side chains. The side chains strongly influence the energetics of heme doming motions that control Fe reactivity, which are easily observed in the experimental signal. (C) 2011 American Institute of Physics. [doi:10.1063/1.3598473]

A copper(II)-hydroperoxo complex, [Cu(Me-6-tren)(OOH)](+) (2), and a copper(II)-cumylperoxo complex, [Cu(Me-6-tren)(OOC(CH3)(2)Ph)](+) (3), were synthesized by reacting [Cu(Me-6-tren)(CH3CN)](2+) (1) with H2O2 and cumyl-OOH, respectively, in the presence of triethylamine. These intermediates, 2 and 3, were successfully characterized by various physicochemical methods such as UV-vis, ESI-MS, resonance Raman and EPR spectroscopies, leading us to propose structures of the Cu(II)-OOR species with a trigonal-bipyramidal geometry. Density functional theory (DFT) calculations provided geometric and electronic configurations of 2 and 3, showing trigonal bipyramidal copper(II)-OOR geometries. These copper(II)-hydroperoxo and -cumylperoxo complexes were inactive in electrophilic and nucleophilic oxidation reactions.

A Cr(V)-oxo complex bearing a macrocyclic TMC ligand, [Cr(V)(TMC)(O)(OCH(3))](2+), was synthesized, isolated, and characterized by various physicochemical methods, including UV-vis, ESI-MS, resonance Raman, EPR and X-ray analysis. The reactivity of the Cr(V)-oxo complex was investigated in C-H and O-H bond activation reactions. The reactivity of a Cr(III)-superoxo complex, [Cr(III) (TMC)(O(2))(Cl)](+), was investigated in O-H bond activation reactions as well. By comparing reactivities of the Cr(III)-superoxo and Cr(V)-oxo complexes under the identical reaction conditions, we were able to demonstrate that the Cr(III)-superoxo complex is more reactive than the Cr(V)-oxo complex in the activation of C-H and O-H bonds. The present results provide strong evidence that under certain circumstances, metal-superoxo species can be an alternative oxidant for high-valent metal-oxo complexes in oxygenation reactions.

Metal-dioxygen adducts are key intermediates detected in the catalytic cycles of dioxygen activation by metalloenzymes and biomimetic compounds. In this study, mononuclear cobalt(III)-peroxo complexes bearing tetraazamacrocyclic ligands, [Co(12-TMC)(O(2))](+) and [Co(13-TMC)(O(2))](+), were synthesized by reacting (Co(12-TMC)(CH(3)CN)](2+) and [Co(13-TMC)(CH(3)CN)](2+), respectively, with H(2)O(2) in the presence of triethylamine. The mononuclear cobalt(III) peroxo intermediates were isolated and characterized by various spectroscopic techniques and X-ray crystallography, and the structural and spectroscopic characterization demonstrated unambiguously that the peroxo ligand is bound in a side-on eta(2) fashion. The O-O bond stretching frequency of [Co(12-TMC)(O(2))](+) and [Co(13-TMC)(O(2))](+) was determined to be 902 cm(-1) by resonance Raman spectroscopy. The structural properties of the CoO(2) core in both complexes are nearly identical; the O-O bond distances of [Co(12-TMC)(O(2))](+) and [Co(13-TMC)(O(2))](+) were 1.4389(17) angstrom and 1.438(6) angstrom, respectively. The cobalt(III)-peroxo complexes showed reactivities in the oxidation of aldehydes and O(2)-transfer reactions. In the aldehyde oxidation reactions, the nucleophilic reactivity of the cobalt peroxo complexes was significantly dependent on the ring size of the macrocyclic ligands, with the reactivity of [Co(13-TMC)(O(2))](+) &gt; [Co(12-TMC)(O(2))](+). In the O(2)-transfer reactions, the cobalt(III)-peroxo complexes transferred the bound peroxo group to a manganese(II) complex, affording the corresponding cobalt(II) and manganese(III)-peroxo complexes. The reactivity of the cobalt-peroxo complexes in O(2)-transfer was also significantly dependent on the ring size of tetraazamacrocycles, and the reactivity order in the O(2)-transfer reactions was the same as that observed in the aldehyde oxidation reactions.

Mn(IV)-peroxo and Mn(V)-oxo corroles were synthesized and characterized with various spectroscopic techniques. The intermediates were directly used in O-O bond cleavage and formation reactions. Upon addition of proton to the Mn(IV)-peroxo corrole, the formation of the Mn(V)-oxo corrole was observed. Interestingly, addition of base to the Mn(V)-oxo corrole afforded the formation of the Mn(IV)-peroxo corrole. Thus, we were able to report the first example of reversible O-O bond cleavage and formation reactions using in situ generated Mn(IV)-peroxo and Mn(V)-oxo corroles.

Nickel(II) complexes supported by a series of tpa {tris[(pyridin-2-yl)methyl]amine} ligand derivatives containing different numbers of phenyl substituent on the 6-position of pyridine donor group (Ph(n)tpa, n = 0-3) have been prepared and structurally characterized. Ligand tpa and its derivatives with one or two 6-Ph substituent(s) (Ph(0)tpa, Ph(1)tpa, and Ph(2)tpa) afforded nickel(II) complexes 1([0]), 1([1]), and 1([2]) with a distorted octahedral geometry, whereas the ligand having three 6-Ph groups (Ph(3)tpa) gave nickel(II) complex 1([3]) with a five-coordinate trigonal bipyramidal structure. All the complexes exhibited a high spin ground state (S = 1). Reactivity of the nickel(H) complexes 1([0]) and 1([1]) toward H2O2 was very poor, whereas 1([2]) and 1([3]) readily gave bis(mu-oxo)dinickel(III) complexes 2121 and 2131, respectively, in the reaction with H2O2 at a low temperature. The bis(mu-oxo)dinickel(III) complexes 2([2]) and 2([3]) gradually decomposed to cause an aromatic hydroxylation reaction of one of the 6-Ph groups of the supporting ligands. The ligand substituent effects on the formation and decomposition processes of the bis(mu-oxo)dinickel(III) complexes are discussed based on the structures and the redox potentials of the starting nickel(II) complexes.

Nitrophorin 4 (NP4) is a heme protein that stores and delivers nitric oxide (NO) through pH-sensitive conformational change. This protein uses the ferric state of a highly ruffled heme to bind NO tightly at low pH and release it at high pH. In this work, the rebinding kinetics of NO and CO to NP4 are investigated as a function of iron oxidation state and the acidity of the environment. The geminate recombination process of NO to ferrous NP4 at both pH 5 and pH 7 is dominated by a single similar to 7 ps kinetic phase that we attribute to the rebinding of NO directly from the distal pocket. The lack of pH dependence explains in part why NP4 cannot use the ferrous state to fulfill its function. The kinetic response of ferric NP4NO shows two distinct phases. The relative geminate amplitude of the slower phase increases dramatically as the pH is raised from 5 to 8. We assign the fast phase of NO rebinding to a conformation of the ferric protein with a closed hydrophobic pocket. The slow phase is assigned to the protein in an open conformation with a more hydrophilic heme pocket environment. Analysis of the ultrafast kinetics finds the equilibrium off-rate of NO to be proportional to the open state population as well as the pH-dependent amplitude of escape from the open pocket. When both factors are considered, the off-rate increases by more than an order of magnitude as the pH changes from 5 to 8. The recombination of CO to ferrous NP4 is observed to have a large nonexponential geminate amplitude with rebinding time scales of similar to 10(-11)-10(-9) s at pH 5 and similar to 10(-10)-10(-8) s at pH 7. The nonexponential CO rebinding kinetics at both pH 5 and pH 7 are accounted for using a simple model that has proven effective for understanding CO binding in a variety of other heme systems (Ye, X.; et al. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 14682).