O2 Binding Research Articles

CO Oxidation Kinetics over Au/TiO2 and Au/Al2O3 Catalysts: Evidence for a Common Water-Assisted Mechanism.

The mechanism of CO oxidation over supported gold catalysts has long been debated, with two prevailing mechanisms dominating the discussion: a water-assisted mechanism and a mechanism involving O-defect sites. In this study, we directly address this debate through a kinetic and mechanistic investigation of the role of water in CO oxidation over Au/TiO2 and Au/Al2O3 catalysts; the results clearly indicate a common water-assisted mechanism to be at work. Water adsorption isotherms were determined with infrared spectroscopy; the extracted equilibrium constant was essentially the same for both catalysts. Added water decreases CO adsorption on Au/TiO2, likely by blocking CO binding sites at the metal-support interface. Reaction kinetics (CO, O2, and H2O reaction orders) were essentially the same for both catalysts, as were measured O-H(D) kinetic isotope effects. These data indicate that the two catalysts operate by essentially the same mechanism under the conditions of these experiments (ambient temperature, significant amounts of water available). A reaction mechanism incorporating the kinetic and thermodynamic data and accounting for different CO and O2/COOH binding sites is proposed. The mechanism and kinetic data are treated with an active site (Michaelis-Menten) approach. This indicated that water adsorption does not significantly affect reaction rate constants, only the number of active sites available at a given water pressure. Extracted water and O2 binding constants are similar on both catalysts and consistent with previous DFT calculations. Water adsorption constants are also similar to independently determined equilibrium constants measured by IR spectroscopy. The likely roles of water, surface carbonates, and oxygen vacancies at the metal-support interface are discussed. The results definitively show that, at least in the presence of added water, O vacancies cannot play an important role in the room-temperature catalysis, and that the water-assisted mechanism is far more consistent with the preponderance of the kinetic data.

A Chiral Ligand Assembly That Confers One-Electron O2 Reduction Activity for a Cu2+ -Selective Metallohydrogel.

The design of functional metallohydrogels is attractive but challenging. A rational approach is introduced for designing functional metallohydrogels using chiral ligands, a phenylalanine derivative with a pyridyl group (l/d-PF). Intriguingly, the as-prepared metallohydrogel exhibits excellent O2 binding and activating properties. Insights into the O2 binding pathway reveals the presence of a novel [(l+d)-PF-Cu3+ -O2- ] species, which can efficiently reduce ferric cytochrome c with the reactive O2- by receiving an electron from reductant ascorbic acid. This study provides helpful instructions for developing new artificial systems with specific functions through the effective combination of chiral ligands with metal ions.

A Chiral Ligand Assembly That Confers One‐Electron O2 Reduction Activity for a Cu2+‐Selective Metallohydrogel

AbstractThe design of functional metallohydrogels is attractive but challenging. A rational approach is introduced for designing functional metallohydrogels using chiral ligands, a phenylalanine derivative with a pyridyl group (l/d‐PF). Intriguingly, the as‐prepared metallohydrogel exhibits excellent O2 binding and activating properties. Insights into the O2 binding pathway reveals the presence of a novel [(l+d)‐PF‐Cu3+‐O2−] species, which can efficiently reduce ferric cytochrome c with the reactive O2− by receiving an electron from reductant ascorbic acid. This study provides helpful instructions for developing new artificial systems with specific functions through the effective combination of chiral ligands with metal ions.

Defined Serum-Free Medium for Bioreactor Culture of an Immortalized Human Erythroblast Cell Line.

Anticipated shortages in donated blood supply have prompted investigation of alternative approaches for in vitro production of red blood cells (RBCs), such as expansion of conditional immortalization erythroid progenitors. However, there is a bioprocessing challenge wherein factors promoting maximal cell expansion and growth-limiting inhibitory factors are yet to be investigated. The authors use an erythroblast cell line (ImEry) derived from immortalizing CD71+CD235a+ erythroblast from adult peripheral blood for optimization of expansion culture conditions. Design of experiments (DOE) is used in media formulation to explore relationships and interactive effects between factors which affect cell expansion. Our in-house optimized medium formulation produced significantly higher cell densities (3.62 ± 0.055) × 106 cells mL-1 , n = 3) compared to commercial formulations (2.07 ± 0.055) × 106 cells mL-1 , n = 3; at 209 h culture). Culture media costs per unit of blood is shown to have a 2.96-3.09 times cost reduction. As a proof of principle for scale up, ImEry are expanded in a half-liter stirred-bioreactor under controlled settings. Growth characteristics, metabolic, and molecular profile of the cells are evaluated. ImEry has identical O2 binding capacity to adult erythroblasts. Amino acid supplementation results in further yield improvements. The study serves as a first step for scaling up erythroblast expansion in controlled bioreactors.

A Breath of Fresh Air: A Genetically Encoded O2 Probe for Direct Mapping and Quantification of Oxygenation Levels in Cells via Fluorescence Lifetime Imaging

Molecular oxygen is an important reporter of metabolic and physiological status at the cellular and tissue level, and its concentration can be used for the evaluation of diseases of various nature (e.g.: cancer, coronary artery disease). The development of accurate and quantitative methods to measure O2 concentration in living cells, tissues and organisms has remained challenging and is subject of intense research. We developed a protein-based, fluorescent oxygen sensor that can be expressed directly in cells to monitor oxygen levels in the cytoplasmic environment. We fused Myoglobin (Mb), a physiological oxygen carrier, with mCherry, a fluorescent protein, to build a fluorescence resonance energy transfer (FRET) pair where Myoglobin acts as a variable dark acceptor. The changes in the spectral properties of Myoglobin upon oxygen binding translate into changes of the FRET-depleted emission intensity of mCherry, and this effect is best detected by monitoring the fluorescence lifetime of the probe. We present in this work the expression and characterization of the Mb-mCherry system in vitro to establish a calibration curve of its response to [O2] and other relevant effectors. Further, by using Fluorescence Lifetime Imaging Microscopy (FLIM), we show that we are able to map the oxygenation level of the probe ex vivo in cells, providing a quantitative description. A genetically encoded oxygen probe based on fluorescence addresses the most common issues with currently available methods to monitor O2 levels in living organisms: data collection speed, spatial resolution and localization within cells and tissues. Customization of the response can be achieved by exploiting the mutations of Mb to tune O2 binding affinity, or the response to external modulators (e.g.: lactate) present in the cell.

Nuclear magnetic resonance-based determination of dioxygen binding sites in protein cavities.

The location and ligand accessibility of internal cavities in cysteine-free wild-type T4 lysozyme was investigated using O2 gas-pressure NMR spectroscopy and molecular dynamics (MD) simulation. Upon increasing the concentration of dissolved O2 in solvent to 8.9 mM, O2 -induced paramagnetic relaxation enhancements (PREs) to the backbone amide and side chain methyl protons were observed, specifically around two cavities in the C-terminal domain. To determine the number of O2 binding sites and their atomic coordinates from the 1/r6 distance dependence of the PREs, we established an analytical procedure using Akaike's Information Criterion, in combination with a grid-search. Two O2 -accessible sites were identified in internal cavities: One site was consistent with the xenon-binding site in the protein in crystal, and the other site was established to be a novel ligand-binding site. MD simulations performed at 10 and 100 mM O2 revealed dioxygen ingress and egress as well as rotational and translational motions of O2 in the cavities. It is therefore suggested that conformational fluctuations within the ground-state ensemble transiently develop channels for O2 association with the internal protein cavities.

A structurally-characterized peroxomanganese(iv) porphyrin from reversible O2 binding within a metal-organic framework.

The role of peroxometal species as reactive intermediates in myriad biological processes has motivated the synthesis and study of analogous molecular model complexes. Peroxomanganese(iv) porphyrin complexes are of particular interest, owing to their potential ability to form from reversible O2 binding, yet have been exceedingly difficult to isolate and characterize in molecular form. Alternatively, immobilization of metalloporphyrin sites within a metal-organic framework (MOF) can enable the study of interactions between low-coordinate metal centers and gaseous substrates, without interference from bimolecular reactions and axial ligation by solvent molecules. Here, we employ this approach to isolate the first rigorously four-coordinate manganese(ii) porphyrin complex and examine its reactivity with O2 using infrared spectroscopy, single-crystal X-ray diffraction, EPR spectroscopy, and O2 adsorption analysis. X-ray diffraction experiments reveal for the first time a peroxomanganese(iv) porphyrin species, which exhibits a side-on, η2 binding mode. Infrared and EPR spectroscopic data confirm the formulation of a peroxomanganese(iv) electronic structure, and show that O2 binding is reversible at ambient temperature, in contrast to what has been observed in molecular form. Finally, O2 gas adsorption measurements are employed to quantify the enthalpy of O2 binding as hads = -49.6(8) kJ mol-1. This enthalpy is considerably higher than in the corresponding Fe- and Co-based MOFs, and is found to increase with increasing reductive capacity of the MII/III redox couple.

An unprecedented dioxygen species revealed by serial femtosecond rotation crystallography in copper nitrite reductase.

Synchrotron-based X-ray structural studies of ligand-bound enzymes are powerful tools to further our understanding of reaction mechanisms. For redox enzymes, it is necessary to study both the oxidized and reduced active sites to fully elucidate the reaction, an objective that is complicated by potential X-ray photoreduction. In the presence of the substrate, this can be exploited to construct a structural movie of the events associated with catalysis. Using the newly developed approach of serial femtosecond rotation crystallography (SF-ROX), an X-ray damage-free structure of the as-isolated copper nitrite reductase (CuNiR) was visualized. The sub-10 fs X-ray pulse length from the SACLA X-ray free-electron laser allowed diffraction data to be collected to 1.6 Å resolution in a 'time-frozen' state. The extremely short duration of the X-ray pulses ensures the capture of data prior to the onset of radiation-induced changes, including radiolysis. Unexpectedly, an O2 ligand was identified bound to the T2Cu in a brand-new binding mode for a diatomic ligand in CuNiRs. The observation of O2 in a time-frozen structure of the as-isolated oxidized enzyme provides long-awaited clear-cut evidence for the mode of O2 binding in CuNiRs. This provides an insight into how CuNiR from Alcaligenes xylosoxidans can function as an oxidase, reducing O2 to H2O2, or as a superoxide dismutase (SOD) since it was shown to have ∼56% of the dismutase activity of the bovine SOD enzyme some two decades ago.

Spin crossover dynamics studies on the thermally activated molecular oxygen binding mechanism on a model copper complex.

The theoretical description of the primary dioxygen (O2) binding and activation step in many copper or iron enzymes, suffers from the intrinsically electronic non-adiabaticity of the spin flip events of the triplet dioxygen molecule (3O2), mediated by spin-orbit couplings. In this work, we presented the early-stage ultrafast spin flip dynamics of O2 binding for a simplified monocopper complex, involving the coupled singlet and triplet electronic states. The on-the-fly trajectory surface hopping (TSH) simulations have identified the dynamical effects that may influence the mode of O2 coordination (end-on vs. side-on), and the electronic structures can be viewed as complexes of molecular O2 with Cu(i) or as Cu(ii)-superoxide compounds. In addition, significant spin flip events are obversed within the sub-picosecond regime. We hope this work may provide complimentary insights on the traditional interpretation of O2 binding on copper complexes and subsequent catalytic reaction mechanisms.

A role of heme side-chains of human hemoglobin in its function revealed by circular dichroism and resonance Raman spectroscopy.

Structural changes of heme side-chains of human adult hemoglobin (Hb A) upon ligand (O2 or CO) dissociation have been studied by circular dichroism (CD) and resonance Raman (RR) spectroscopies. We point out the occurrence of appreciable deformation of heme side-chains like vinyl and propionate groups prior to the out-of-plane displacement of heme iron. Referring to the recent fine resolved crystal structure of Hb A, the deformations of heme side-chains take place only in the β subunits. However, these changes are not observed in the isolated β chain (β4 homotetramer) and, therefore, are associated with the α-β inter-subunit interactions. For the communications between α and β subunits in Hb A regarding signals of ligand dissociation, possible routes are proposed on the basis of the time-resolved absorption, CD, MCD (magnetic CD), and RR spectroscopies. Our finding of the movements of heme side-chains would serve as one of the clues to solve the cooperative O2 binding mechanism of Hb A.

Ligand-Directed Reactivity in Dioxygen and Water Binding to cis-[Pd(NHC)2(η2-O2)

Reaction of [Pd(IPr)2] (IPr = 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene) and O2 leads to the surprising discovery that at low temperature the initial reaction product is a highly labile peroxide complex cis-[Pd(IPr)2(η2-O2)]. At temperatures ≳ -40 °C, cis-[Pd(IPr)2(η2-O2)] adds a second O2 to form trans-[Pd(IPr)2(η1-O2)2]. Squid magnetometry and EPR studies yield data that are consistent with a singlet diradical ground state with a thermally accessible triplet state for this unique bis-superoxide complex. In addition to reaction with O2, cis-[Pd(IPr)2(η2-O2)] reacts at low temperature with H2O in methanol/ether solution to form trans-[Pd(IPr)2(OH)(OOH)]. The crystal structure of trans-[Pd(IPr)2(OOH)(OH)] is reported. Neither reaction with O2 nor reaction with H2O occurs under comparable conditions for cis-[Pd(IMes)2(η2-O2)] (IMes = 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene). The increased reactivity of cis-[Pd(IPr)2(η2-O2)] is attributed to the enthalpy of binding of O2 to [Pd(IPr)2] (-14.5 ± 1.0 kcal/mol) that is approximately one-half that of [Pd(IMes)2] (-27.9 ± 1.5 kcal/mol). Computational studies identify the cause as interligand repulsion forcing a wider C-Pd-C angle and tilting of the NHC plane in cis-[Pd(IPr)2(η2-O2)]. Arene-arene interactions are more favorable and serve to further stabilize cis-[Pd(IMes)2(η2-O2)]. Inclusion of dispersion effects in DFT calculations leads to improved agreement between experimental and computational enthalpies of O2 binding. A complete reaction diagram is constructed for formation of trans-[Pd(IPr)2(η1-O2)2] and leads to the conclusion that kinetic factors inhibit formation of trans-[Pd(IMes)2(η1-O2)2] at the low temperatures at which it is thermodynamically favored. Failure to detect the predicted T-shaped intermediate trans-[Pd(NHC)2(η1-O2)] for either NHC = IMes or IPr is attributed to dynamic effects. A partial potential energy diagram for initial binding of O2 is constructed. A range of low-energy pathways at different angles of approach are present and blur the distinction between pure "side-on" or "end-on" trajectories for oxygen binding.

Nickel‐substituted iron‐dependent cysteine dioxygenase: Implications for the dioxygenation activity of nickel model compounds

Reported here is a density functional theory study on the ability of Ni‐substituted iron‐dependent cysteine dioxygenase (CDO) to catalyze the oxidation of cysteine to cysteine sulfinic acid. The first steps of the commonly accepted mechanism for CDO, the O2 activation mechanism, suggests the binding of O2 to the metal ion (where redox isomerism takes place converting O2 to ) followed by the attack of the distal oxygen atom on the cysteine sulfur—in line with most previous evidence. An alternative mechanism entailing the attack of the cysteine sulfur on the proximal oxygen atom of the dioxygen moiety to form a persulfenate intermediate without any redox exchange between the metal ion and the O2 ligand, is supported by an X‐ray crystal structure showing a CDO with a bound cysteine persulfenate, and also supported by data on the oxidation of thiols catalyzed by Ni(II) compounds. Our results show that the O2 activation mechanism with a Ni‐substituted active site follows the same pattern as native CDOs albeit with much higher energy barriers for the formation of the intermediates suggesting that the reaction might not be biologically feasible. Conversely, the immediate cleavage of the persulfenate SO bond in the alternative mechanism suggests that cysteine persulfenate might not be a true intermediate in catalytic cycle of CDOs.

Coordination and redox state–dependent structural changes of the heme-based oxygen sensor AfGcHK associated with intraprotein signal transduction

The heme-based oxygen sensor histidine kinase AfGcHK is part of a two-component signal transduction system in bacteria. O2 binding to the Fe(II) heme complex of its N-terminal globin domain strongly stimulates autophosphorylation at His183 in its C-terminal kinase domain. The 6-coordinate heme Fe(III)-OH- and -CN- complexes of AfGcHK are also active, but the 5-coordinate heme Fe(II) complex and the heme-free apo-form are inactive. Here, we determined the crystal structures of the isolated dimeric globin domains of the active Fe(III)-CN- and inactive 5-coordinate Fe(II) forms, revealing striking structural differences on the heme-proximal side of the globin domain. Using hydrogen/deuterium exchange coupled with mass spectrometry to characterize the conformations of the active and inactive forms of full-length AfGcHK in solution, we investigated the intramolecular signal transduction mechanisms. Major differences between the active and inactive forms were observed on the heme-proximal side (helix H5), at the dimerization interface (helices H6 and H7 and loop L7) of the globin domain and in the ATP-binding site (helices H9 and H11) of the kinase domain. Moreover, separation of the sensor and kinase domains, which deactivates catalysis, increased the solvent exposure of the globin domain-dimerization interface (helix H6) as well as the flexibility and solvent exposure of helix H11. Together, these results suggest that structural changes at the heme-proximal side, the globin domain-dimerization interface, and the ATP-binding site are important in the signal transduction mechanism of AfGcHK. We conclude that AfGcHK functions as an ensemble of molecules sampling at least two conformational states.

Probing Ligand Exchange in the P450 Enzyme CYP121 from Mycobacterium tuberculosis: Dynamic Equilibrium of the Distal Heme Ligand as a Function of pH and Temperature.

CYP121 is a cytochrome P450 enzyme from Mycobacterium tuberculosis that catalyzes the formation of a C-C bond between the aromatic groups of its cyclodityrosine substrate (cYY). The crystal structure of CYP121 in complex with cYY reveals that the solvent-derived ligand remains bound to the ferric ion in the enzyme-substrate complex. Whereas in the generally accepted P450 mechanism, binding of the primary substrate in the active-site triggers the release of the solvent-derived ligand, priming the metal center for reduction and subsequent O2 binding. Here we employed sodium cyanide to probe the metal-ligand exchange of the enzyme and the enzyme-substrate complex. The cyano adducts were characterized by UV-vis, EPR, and ENDOR spectroscopies and X-ray crystallography. A 100-fold increase in the affinity of cyanide binding to the enzyme-substrate complex over the ligand-free enzyme was observed. The crystal structure of the [CYP121(cYY)CN] ternary complex showed a rearrangement of the substrate in the active-site, when compared to the structure of the binary [CYP121(cYY)] complex. Transient kinetic studies showed that cYY binding resulted in a lower second-order rate constant (kon(CN)) but a much more stable cyanide adduct with 3 orders of magnitude slower koff(CN) rate. A dynamic equilibrium between multiple high- and low-spin species for both the enzyme and enzyme-substrate complex was also observed, which is sensitive to changes in both pH and temperature. Our data reveal the chemical and physical properties of the solvent-derived ligand of the enzyme, which will help to understand the initial steps of the catalytic mechanism.

Functional diversification of sea lamprey globins in evolution and development

Agnathans have a globin repertoire that markedly differs from that of jawed (gnathostome) vertebrates. The sea lamprey (Petromyzon marinus) harbors at least 18 hemoglobin, two myoglobin, two globin X, and one cytoglobin genes. However, agnathan hemoglobins and myoglobins are not orthologous to their cognates in jawed vertebrates. Thus, blood-based O2 transport and muscle-based O2 storage proteins emerged twice in vertebrates from a tissue-globin ancestor. Notably, the sea lamprey displays three switches in hemoglobin expression in its life cycle, analogous to hemoglobin switching in vertebrates. To study the functional changes associated with the evolution and ontogenesis of distinct globin types, we determined O2 binding equilibria, type of quaternary assembly, and nitrite reductase enzymatic activities of one adult (aHb5a) and one embryonic/larval hemoglobin (aHb6), myoglobin (aMb1) and cytoglobin (Cygb) of the sea lamprey. We found clear functional differentiation among globin types expressed at different developmental stages and in different tissues. Cygb and aMb1 have high O2 affinity and nitrite reductase activity, while the two hemoglobins display low O2 affinity and nitrite reductase activity. Cygb and aHb6 but not aHb5a show cooperative O2 binding, correlating with increased stability of dimers, as shown by gel filtration and molecular modeling. The high O2-affinity and the lack of cooperativity confirm the identity of the sea lamprey aMb1 as O2 storage protein of the muscle. The dimeric structure and O2-binding properties of sea lamprey and mammalian Cygb were very similar, suggesting a conservation of function since their divergence around 500million years ago.

Heterogeneity between Two α Subunits of α2β2 Human Hemoglobin and O2 Binding Properties: Raman, 1H Nuclear Magnetic Resonance, and Terahertz Spectra.

Following a previous detailed investigation of the β subunit of α2β2 human adult hemoglobin (Hb A), this study focuses on the α subunit by using three natural valency hybrid α(Fe2+-deoxy/O2)β(Fe3+) hemoglobin M (Hb M) in which O2 cannot bind to the β subunit: Hb M Hyde Park (β92His → Tyr), Hb M Saskatoon (β63His → Tyr), and Hb M Milwaukee (β67Val → Glu). In contrast with the β subunit that exhibited a clear correlation between O2 affinity and Fe2+-His stretching frequencies, the Fe2+-His stretching mode of the α subunit gave two Raman bands only in the T quaternary structure. This means the presence of two tertiary structures in α subunits of the α2β2 tetramer with T structure, and the two structures seemed to be nondynamical as judged from terahertz absorption spectra in the 5-30 cm-1 region of Hb M Milwaukee, α(Fe2+-deoxy)β(Fe3+). This kind of heterogeneity of α subunits was noticed in the reported spectra of a metal hybrid Hb A like α(Fe2+-deoxy)β(Co2+) and, therefore, seems to be universal among α subunits of Hb A. Unexpectedly, the two Fe-His frequencies were hardly changed with a large alteration of O2 affinity by pH change, suggesting no correlation of frequency with O2 affinity for the α subunit. Instead, a new Fe2+-His band corresponding to the R quaternary structure appeared at a higher frequency and was intensified as the O2 affinity increased. The high-frequency counterpart was also observed for a partially O2-bound form, α(Fe2+-deoxy)α(Fe2+-O2)β(Fe3+)β(Fe3+), of the present Hb M, consistent with our previous finding that binding of O2 to one α subunit of T structure α2β2 tetramer changes the other α subunit to the R structure.

Dioxygen Activation and O-O Bond Formation Reactions by Manganese Corroles.

Activation of dioxygen (O2) in enzymatic and biomimetic reactions has been intensively investigated over the past several decades. More recently, O-O bond formation, which is the reverse of the O2-activation reaction, has been the focus of current research. Herein, we report the O2-activation and O-O bond formation reactions by manganese corrole complexes. In the O2-activation reaction, Mn(V)-oxo and Mn(IV)-peroxo intermediates were formed when Mn(III) corroles were exposed to O2 in the presence of base (e.g., OH-) and hydrogen atom (H atom) donor (e.g., THF or cyclic olefins); the O2-activation reaction did not occur in the absence of base and H atom donor. Moreover, formation of the Mn(V)-oxo and Mn(IV)-peroxo species was dependent on the amounts of base present in the reaction solution. The role of the base was proposed to lower the oxidation potential of the Mn(III) corroles, thereby facilitating the binding of O2 and forming a Mn(IV)-superoxo species. The putative Mn(IV)-superoxo species was then converted to the corresponding Mn(IV)-hydroperoxo species by abstracting a H atom from H atom donor, followed by the O-O bond cleavage of the putative Mn(IV)-hydroperoxo species to form a Mn(V)-oxo species. We have also shown that addition of hydroxide ion to the Mn(V)-oxo species afforded the Mn(IV)-peroxo species via O-O bond formation and the resulting Mn(IV)-peroxo species reverted to the Mn(V)-oxo species upon addition of proton, indicating that the O-O bond formation and cleavage reactions between the Mn(V)-oxo and Mn(IV)-peroxo complexes are reversible. The present study reports the first example of using the same manganese complex in both O2-activation and O-O bond formation reactions.

Pathways and Thermodynamics of Oxygen Diffusion in [FeFe]-Hydrogenase.

The H2 production potential of [FeFe]-hydrogenase, a hydrogen-producing enzyme from green algae, is reported to be promising for economical and large-scale production of H2 as an alternative source of renewable energy. The production of hydrogen takes place at the catalytic center buried in the enzyme core. Unfortunately, binding of O2 to the catalytic center of the enzyme irreversibly inactivates it, essentially blocking hydrogen production. Therefore, a better understanding of the mechanism of O2 entry/exit is necessary to develop strategies for designing oxygen-tolerant enzymes. In this work, we investigated the pathways and diffusion channels of O2 gas in this hydrogenase. Through exhaustive mapping of oxygen-diffusion channels, we computed a full thermodynamic map of preferred binding locations of O2 gas within the enzyme interior, which showed that O2 can enter and exit the enzyme through multiple pathways along which are key residues that are known to perturb rates of O2 binding. The global minimum in the free-energy landscape is located near the H-cluster, a key metallic center within the enzyme. Along O2 diffusion channels, we further identified several residues that could be potential candidates for mutations to increase the oxygen tolerance of [FeFe]-hydrogenase.

Mechanism of the Nitric Oxide Dioxygenase Reaction of Mycobacterium tuberculosis Hemoglobin N.

Many globins convert •NO to innocuous NO3- through their nitric oxide dioxygenase (NOD) activity. Mycobacterium tuberculosis fights the oxidative and nitrosative stress imposed by its host (the toxic effects of O2•- and •NO species and their OONO- and •NO2 derivatives) through the action of truncated hemoglobin N (trHbN), which catalyzes the NOD reaction with one of the highest rates among globins. The general NOD mechanism comprises the following steps: binding of O2 to the heme, diffusion of •NO into the heme pocket and formation of peroxynitrite (OONO-), isomerization of OONO-, and release of NO3-. Using quantum mechanics/molecular mechanics free-energy calculations, we show that the NOD reaction in trHbN follows a mechanism in which heme-bound OONO- undergoes homolytic cleavage to give FeIV═O2- and the •NO2 radical but that these potentially harmful intermediates are short-lived and caged by the heme pocket residues. In particular, the simulations show that Tyr33(B10) side chain is shielded from FeIV═O2- and •NO2 (and protected from irreversible oxidation and nitration) by forming stable hydrogen bonds with Gln58(E11) side chain and Leu54(E7) backbone. Aromatic residues Phe46(CD1), Phe32(B9), and Tyr33(B10) promote NO3- dissociation via C-H···O bonding and provide stabilizing interactions for the anion along its egress route.

PP.21.02] MENTAL STRESS, PH AND PCO2 AND SYSTOLIC HYPERTENSION

Objective: Resilence is known to be a major risk factor for the development of primary hypertension. Persisting overcompensation during mental irritation increases pH values and oxygen binding in blood samples by respiratory overcompensation. Is this metabolic effect dependent on blood pressure values? Design and method: 23 healthy young officer trainees were tested for pCO2, O2 saturation, pH values (PhOX-M, TECOM, Austria) and blood pressure values (Beurer, Ulm, Germany) during the sympatho-adrenal anticipation phase before their first parachute jump. Both, the entire group as well as two subgroups, consisting of systolic blood pressure values above average and below average (normal values) have been investigated. Results: The relation pH/pCO2, indicative of breathing frequency and acid management shows a rather weak negative linear correlation in the entire group (r = 0.329). The normal systolic blood pressure group shows no correlation at all, the high systolic blood pressure group developed a highly significant negative correlation of pH/CO2 (r = 0.656, p < 0.001). At the same time, O2 saturation increases with increasingly alkaline blood and decreasing pCO2 (p < 0.05). Conclusions: Mental stimulation increases blood pH by respiratory overcompensation and thereby increases O2 binding. Consequently, less O2 reaches e.g. heart tissue at higher demand during a sympatho-adrenal anticipation phase, which is by no means uncommom during day-to-day exertions. This symptom is much less significant at normal blood pressure values, but highly significant at higher systolic blood pressure values. Therefore, increased systolic blood pressure values institute an integral part of significantly negative metabolic effects upon the heart during even commonplace mental stimulation or irritation.