O2 Binding Research Articles

Non‐Coordinative Binding of O2 at the Active Center of a Copper‐Dependent Enzyme

AbstractMolecular oxygen (O2) is a sustainable oxidation reagent. O2 is strongly oxidizing but kinetically stable and its final reaction product is water. For these reasons learning how to activate O2 and how to steer its reactivity along desired reaction pathways is a longstanding challenge in chemical research.[1] Activation of ground‐state diradical O2 can occur either via conversion to singlet oxygen or by one‐electron reduction to superoxide. Many enzymes facilitate activation of O2 by direct fomation of a metal‐oxygen coordination complex concomitant with inner sphere electron transfer. The formylglycine generating enzyme (FGE) is an unusual mononuclear copper enzyme that appears to follow a different strategy. Atomic‐resolution crystal structures of the precatalytic complex of FGE demonstrate that this enzyme binds O2 juxtaposed, but not coordinated to the catalytic CuI. Isostructural complexes that contain AgI instead of CuI or nitric oxide instead of O2 confirm that formation of the initial oxygenated complex of FGE does not depend on redox activity. A stepwise mechanism that decouples binding and activation of O2 is unprecedented for metal‐dependent oxidases, but is reminiscent of flavin‐dependent enzymes.

Non-Coordinative Binding of O2 at the Active Center of a Copper-Dependent Enzyme.

Molecular oxygen (O2 ) is a sustainable oxidation reagent. O2 is strongly oxidizing but kinetically stable and its final reaction product is water. For these reasons learning how to activate O2 and how to steer its reactivity along desired reaction pathways is a longstanding challenge in chemical research.[1] Activation of ground-state diradical O2 can occur either via conversion to singlet oxygen or by one-electron reduction to superoxide. Many enzymes facilitate activation of O2 by direct fomation of a metal-oxygen coordination complex concomitant with inner sphere electron transfer. The formylglycine generating enzyme (FGE) is an unusual mononuclear copper enzyme that appears to follow a different strategy. Atomic-resolution crystal structures of the precatalytic complex of FGE demonstrate that this enzyme binds O2 juxtaposed, but not coordinated to the catalytic CuI . Isostructural complexes that contain AgI instead of CuI or nitric oxide instead of O2 confirm that formation of the initial oxygenated complex of FGE does not depend on redox activity. A stepwise mechanism that decouples binding and activation of O2 is unprecedented for metal-dependent oxidases, but is reminiscent of flavin-dependent enzymes.

An Engineered Glutamate in Biosynthetic Models of Heme-Copper Oxidases Drives Complete Product Selectivity by Tuning the Hydrogen-Bonding Network.

Efficiently carrying out the oxygen reduction reaction (ORR) is critical for many applications in biology and chemistry, such as bioenergetics and fuel cells, respectively. In biology, this reaction is carried out by large, transmembrane oxidases such as heme-copper oxidases (HCOs) and cytochrome bd oxidases. Common to these oxidases is the presence of a glutamate residue next to the active site, but its precise role in regulating the oxidase activity remains unclear. To gain insight into its role, we herein report that incorporation of glutamate next to a designed heme-copper center in two biosynthetic models of HCOs improves O2 binding affinity, facilitates protonation of reaction intermediates, and eliminates release of reactive oxygen species. High-resolution crystal structures of the models revealed extended, water-mediated hydrogen-bonding networks involving the glutamate. Electron paramagnetic resonance of the cryoreduced oxy-ferrous centers at cryogenic temperature followed by thermal annealing allowed observation of the key hydroperoxo intermediate that can be attributed to the hydrogen-bonding network. By demonstrating these important roles of glutamate in oxygen reduction biochemistry, this work offers deeper insights into its role in native oxidases, which may guide the design of more efficient artificial ORR enzymes or catalysts for applications such as fuel cells.

Density Functional Theory Study of Oxygen Reduction on Graphene and Platinum Surfaces of Pt–Graphene Hybrids

Based on recent studies, an epitaxial monolayer of Pt on graphene yielded an enhanced catalytic activity with unprecedented stability resulting in an improved catalyst lifetime. We showed that the origin of this stability is due to strong Pt–C covalency and unique Pt and graphene surface morphologies compared to precedent physically binding metal–graphene surfaces or impurity-doped graphene catalysts. In this study, the oxygen reduction reaction (ORR) mechanisms on both surfaces are investigated using density functional theory for the first time. This two-dimensional catalyst possesses two reactive surfaces for ORR: one is the Pt surface and the other is the graphene surface. The graphene surface is found to have an undulated morphology from the sp2–sp3 alternating hybridization of carbon atoms in graphene, which are generated due to the covalent bond formation with Pt atoms. The sp2 region of graphene provides active reaction sites for the ORR, and all active sites on the Pt surface are characterized from the Pt–C(sp3) bonds. It is revealed that O2 adsorption takes place via a dissociative chemisorption mechanism on both catalytic surfaces. Compared to the Pt surface, ORR on the graphene surface would be more facile due to a weaker O2 binding energy and a lower overpotential. This study shows that a Pt–graphene hybrid system without defects or impurities on graphene is capable of exhibiting an electrochemical catalytic activity.

Weak O2 binding and strong H2O2 binding at the non-heme diiron center of trypanosome alternative oxidase

Alternative oxidase (AOX) catalyzes the four-electron reduction of dioxygen to water as an additional terminal oxidase, and the catalytic reaction is critical for the parasite to survive in its bloodstream form. Recently, the X-ray crystal structure of trypanosome alternative oxidase (TAO) complexed with ferulenol was reported and the molecular structure of the non-heme diiron center was determined. The binding of O2 was a unique side-on type compared to other iron proteins. In order to characterize the O2 binding state of TAO, the O2 binding states were searched at a quantum mechanics/molecular mechanics (QM/MM) theoretical level in the present study. We found that the most stable O2 binding state is the end-on type, and the binding states of the side-on type are higher in energy. Based on the binding energies and electronic structure analyses, O2 binds very weakly to the TAO iron center (ΔE =6.7 kcal mol−1) in the electronic state of Fe(II)…OO, not in the suggested charge transferred state such as the superoxide state (Fe(III)OO· –) as seen in hemerythrin. Coordination of other ligands such as water, Cl−, CN−, CO, N3− and H2O2 was also examined, and H2O2 was found to bind most strongly to the Fe(II) site by ΔE = 14.0 kcal mol−1. This was confirmed experimentally through the measurement of ubiquinol oxidase activity of TAO and Cryptosporidium parvum AOX which was found to be inhibited by H2O2 in a dose-dependent and reversible manner.

Modulating the crystallinity of boron nitride for propane oxidative dehydrogenation

In this work, we optimized the catalytic performances of h-BN in propane oxidative dehydrogenation by modulating the catalyst crystallinity. The propane conversion rate per unit specific surface area of the catalyst (-mol C3H8/m2/s) was improved by almost ten times by modulating the crystallinity of BN catalyst, gaining a propylene productivity of about 3.10 μmol g cat−1 s−1. The reaction activity presented a negative relationship with the specific surface area but positively related to the crystallinity of BN. High degree of crystallinity is proved to be beneficial for the binding of O2 to BN by the O2-TPD results and zeta potentials of BN-T, which could probably reduce the energy barrier for the activation of O2 and the formation of active sites. Additionally, the increase in propane conversion with the increase of reaction temperature tends to be gentle when the reaction temperature is above 530 °C, and it is almost equal to zero for O2/C3H8 value increasing from 1.30 to 2.00, indicating an energy-efficient and economical route to convert C3H8 over BN catalyst with high crystallinity.

Spin-Crossover Effects in Reversible O2 Binding on a Dinuclear Cobalt(II) Complex

Reversible and selective binding of O2 has been demonstrated previously in a series of experiments with di-nuclear and tetra-nuclear cobalt(II) complexes, including materials containing the redox-a...

Influence of Cyano Substituents on the Electron Density and Catalytic Activity Towards the Oxygen Reduction Reaction for Iron Phthalocyanine

Iron(II) 2,3,9,10,16,17,23,24-octa(cyano)phthalocyanine (OCNFePc), was tested as a catalyst for the oxygen reduction reaction (ORR) adsorbed on carbon nanotubes. The composite was analyzed spectroscopically and electrochemically characterized at pH 13 and pH 1. The composite showed close to 4 electron processes at pH 13. Computational analysis indicates that the O2 molecule binds end-on to the metal center and that the dioxygen molecule is dissociated on both the Fe metal center and the corral ring. An Analysis of the molecular electrostatic potential confirms the behavior of cyano residues as electron-withdrawing moieties in OCNFePc. As a result of its catalytic behavior and theoretical analysis of its O2 binding energy, OCNFePc was placed in a high position on a volcano correlation of similar phthalocyanine composites.

Intramolecular Electrostatic Effects on O2, CO2, and Acetate Binding to a Cationic Iron Porphyrin.

Noncovalent electrostatic interactions are important in many biological and chemical reactions, especially those that involve charged intermediates. There has been a growing interest in using electrostatic ligand designs-placing charges in the second coordination sphere-to improve molecular reactivity, catalysis, and electrocatalysis. For instance, an iron porphyrin bearing four cationic ortho-trimethylanilinium groups, Fe(o-TMA), has been reported to be an exceptional electrocatalyst for both the carbon dioxide reduction reaction (CO2RR) and the oxygen reduction reaction (ORR). These reactions involve many different steps, and it is not evident which steps are affected by the four positive charges, or why. By comparing Fe(o-TMA) with the related iron-tetraphenylporphyrin, this work examines how covalently positioned charged groups affect substrate binding and other key pre-equilibria of both the ORR and CO2RR, specifically acetate, dioxygen, and carbon dioxide binding. This study is among the first to directly measure the effects of electrostatics on ligand-binding. The results show that adding electrostatic groups to a catalyst design often results in a complex interplay of multiple effects, including changes in pre-equilibria prior to substrate binding, combinations of through-space and inductive contributions, and effects of ionic strength and solution dielectric. The inverse half-order dependence of binding constant on ionic strength is proposed as a clear marker for an electrostatic effect. The conclusions provide guidance for the increasingly popular electrostatic ligand designs in catalysis and other reactivity.

Red blood cell carbonic anhydrase mediates oxygen delivery via the Root effect in red drum.

Oxygen (O2) and carbon dioxide (CO2) transport are tightly coupled in many fishes as a result of the presence of Root effect hemoglobins (Hb), whereby reduced pH reduces O2 binding even at high O2 tensions. Red blood cell carbonic anhydrase (RBC CA) activity limits the rate of intracellular acidification, yet its role in O2 delivery has been downplayed. We developed an in vitro assay to manipulate RBC CA activity while measuring Hb-O2 offloading following a physiologically relevant CO2-induced acidification. RBC CA activity in red drum (Sciaenops ocellatus) was inhibited with ethoxzolamide by 53.7±0.5%, which prompted a significant reduction in O2 offloading rate by 54.3±5.4% (P=0.0206, two-tailed paired t-test; n=7). Conversely, a 2.03-fold increase in RBC CA activity prompted a 2.14-fold increase in O2 offloading rate (P<0.001, two-tailed paired t-test; n=8). This approximately 1:1 relationship between RBC CA activity and Hb-O2 offloading rate coincided with a similar allometric scaling exponent for RBC CA activity and maximum metabolic rate. Together, our data suggest that RBC CA is rate limiting for O2 delivery in red drum.

Dioxygen Binding to all 3d, 4d, and 5d Transition Metals from Coupled-Cluster Theory.

Understanding how transition metals bind and activate dioxygen (O2 ) is limited by experimental and theoretical uncertainties, making accurate quantum mechanical descriptors of interest. Here we report coupled-cluster CCSD(T) energies with large basis sets and vibrational and relativistic corrections for 160 3d, 4d, and 5d metal-O2 systems. We define four reaction energies (120 in total for the 30 metals) that quantify O-O activation and reveal linear relationships between metal-oxygen and O-O binding energies. The CCSD(T) data can be combined with thermochemical cycles to estimate chemisorption and physisorption energies for each metal from metal oxide embedding energies, in good correlation with atomization enthalpies (R2 =0.75). Spin-geometry variations can break the linearities, of interest to circumventing the Sabatier principle. Pt, Pd, Co, and Fe form a distinct group with the weakest O2 binding. R2 up to 0.84 between surface adsorption energies and our energies for MO2 systems indicate relevance also to real catalytic systems.

Enhancing water splitting via weakening H2 and O2 adsorption on NiCo-LDH@CdS due to interstitial nitrogen doping: A close look at the mechanism of electron transfer

This paper is a report on the development of a convenient approach to fabricating a very efficient hybrid photoelectrocatalyst for water splitting. This photoelectrocatalyst consists of nickel–cobalt layered double hydroxide as the core, cadmium sulfide as the shell, and nitrogen, hence [email protected] For the electrocatalytic activity to be improved, the H2 and O2 binding energy needs to be weakened. The interstitial nitrogen doping on [email protected] can increase electrocatalytic activity to a great extent. [email protected] nanoparticles are obtained by subjecting to nitriding the [email protected] electrode coated with polyvinylpyrrolidone nanosheets. This electrode has a large specific surface area, allows fast transfer of electrons, and exhibits long-term stability. The experimental results presented in this paper reveal that interstitial nitrogen doping largely reduces H2 and O2 binding energy and lowers the activation barrier for the formation and splitting of water.

Spin-inversion mechanisms in O2 binding to a model heme compound: A perspective from nonadiabatic wave packet calculations.

Spin-inversion dynamics in O2 binding to a model heme complex, which consisted of Fe(II)-porphyrin and imidazole, were studied using nonadiabatic wave packet dynamics calculations. We considered three active nuclear degrees of freedom in the dynamics, including the motions along the Fe-O distance, Fe-O-O angle, and Fe out-of-plane distance. Spin-free potential energy surfaces for the singlet, triplet, quintet, and septet states were developed using density functional theory calculations, and spin-orbit coupling elements were obtained from CASSCF-level electronic structure calculations. The spin-inversion mainly occurred between the singlet state and one of the triplet states due to large spin-orbit couplings and the contributions of other states were extremely small. The present quantum dynamics calculations suggested that the narrow crossing region model plays a dominant role in the O2 binding dynamics. In addition, the one-dimensional Landau-Zener model underestimated the nonadiabatic transition probability.

Catalytic Mechanism of Aromatic Nitration by Cytochrome P450 TxtE: Involvement of a Ferric-Peroxynitrite Intermediate

The cytochromes P450 are heme-dependent enzymes that catalyze many vital reaction processes in the human body related to biodegradation and biosynthesis. They typically act as mono-oxygenases; however, the recently discovered P450 subfamily TxtE utilizes O2 and NO to nitrate aromatic substrates such as L-tryptophan. A direct and selective aromatic nitration reaction may be useful in biotechnology for the synthesis of drugs or small molecules. Details of the catalytic mechanism are unknown, and it has been suggested that the reaction should proceed through either an iron(III)-superoxo or an iron(II)-nitrosyl intermediate. To resolve this controversy, we used stopped-flow kinetics to provide evidence for a catalytic cycle where dioxygen binds prior to NO to generate an active iron(III)-peroxynitrite species that is able to nitrate l-Trp efficiently. We show that the rate of binding of O2 is faster than that of NO and also leads to l-Trp nitration, while little evidence of product formation is observed from the iron(II)-nitrosyl complex. To support the experimental studies, we performed density functional theory studies on large active site cluster models. The studies suggest a mechanism involving an iron(III)-peroxynitrite that splits homolytically to form an iron(IV)-oxo heme (Compound II) and a free NO2 radical via a small free energy of activation. The latter activates the substrate on the aromatic ring, while compound II picks up the ipso-hydrogen to form the product. The calculations give small reaction barriers for most steps in the catalytic cycle and, therefore, predict fast product formation from the iron(III)-peroxynitrite complex. These findings provide the first detailed insight into the mechanism of nitration by a member of the TxtE subfamily and highlight how the enzyme facilitates this novel reaction chemistry.

Unveiling VIV O2+ Binding Modes to Human Serum Albumins by an Integrated Spectroscopic-Computational Approach.

Human serum albumin (HSA) is involved in the transport of metal ions and potential metallodrugs. Depending on the metal, several sites are available, among which are N-terminal (NTS) and multi-metal binding sites (MBS). Despite the large number of X-ray determinations for albumins, only one structure with Zn2+ is available. In this work, the binding to HSA of the VIV O2+ ion was studied by an integrated approach based on spectroscopic and computational methods, which allowed the systems to be characterized even in the absence of X-ray analysis. The behavior depends on the type of albumin, defatted (HSAd ) or fatted (HSAf ). With HSAd 'primary' and 'secondary' sites were revealed, NTS with (His3, His9, Asp13, Asp255) and MBS with (His67, His247, Asp249, Asn99 or H2 O); with increasing the ratio VIV O2+ /HSAd , 'tertiary' sites, with one His-N and other donors (Asp/Glu-O or carbonyl-O) are populated. With HSAf , fatty acids (FAs) cause a rotation of the subdomains IA and IIA, which results in the formation of a dinuclear ferromagnetic adduct (VIV O)2 D (HSAf ) with a μ1,1 -Asp249 and the binding of His247, Glu100, Glu252, and His67 or Asn99. FAs hinder also the binding of VIV O2+ to the MBS.

Structural Studies of the Methylosinus trichosporium OB3b Soluble Methane Monooxygenase Hydroxylase and Regulatory Component Complex Reveal a Transient Substrate Tunnel.

The metalloenzyme soluble methane monooxygenase (sMMO) consists of hydroxylase (sMMOH), regulatory (MMOB), and reductase components. When sMMOH forms a complex with MMOB, the rate constants are greatly increased for the sequential access of O2, protons, and CH4 to an oxygen-bridged diferrous metal cluster located in the buried active site. Here, we report high-resolution X-ray crystal structures of the diferric and diferrous states of both sMMOH and the sMMOH:MMOB complex using the components from Methylosinus trichosporium OB3b. These structures are analyzed for O2 access routes enhanced when the complex forms. Previously reported, lower-resolution structures of the sMMOH:MMOB complex from the sMMO of Methylococcus capsulatus Bath revealed a series of cavities through sMMOH postulated to serve as the O2 conduit. This potential role is evaluated in greater detail using the current structures. Additionally, a search for other potential O2 conduits in the M. trichosporium OB3b sMMOH:MMOB complex revealed a narrow molecular tunnel, termed the W308-tunnel. This tunnel is sized appropriately for O2 and traverses the sMMOH-MMOB interface before accessing the active site. The kinetics of reaction of O2 with the diferrous sMMOH:MMOB complex in solution show that use of the MMOB V41R variant decreases the rate constant for O2 binding >25000-fold without altering the component affinity. The location of Val41 near the entrance to the W308-tunnel is consistent with the tunnel serving as the primary route for the transfer of O2 into the active site. Accordingly, the crystal structures show that formation of the diferrous sMMOH:MMOB complex restricts access through the chain of cavities while opening the W308-tunnel.

Synthesis of Recombinant Human Hemoglobin With NH2 -Terminal Acetylation in Escherichia coli.

The development of new technologies for the efficient expression of recombinant hemoglobin (rHb) is of interest for experimental studies of protein biochemistry and the development of cell-free blood substitutes in transfusion medicine. Expression of rHb in Escherichia coli host cells has numerous advantages, but one disadvantage of using prokaryotic systems to express eukaryotic proteins is that they are incapable of performing post-translational modifications such as NH2 -terminal acetylation. One possible solution is to coexpress additional enzymes that can perform the necessary modifications in the host cells. Here, we report a new method for synthesizing human rHb with proper NH2 -terminal acetylation. Mass spectrometry experiments involving native and recombinant human Hb confirmed the efficacy of the new technique in producing correctly acetylated globin chains. Finally, functional experiments provided insights into the effects of NH2 -terminal acetylation on O2 binding properties. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Gene synthesis and cloning the cassette to the expression plasmid Basic Protocol 2: Selection of E. coli expression strains for coexpression Basic Protocol 3: Large-scale recombinant hemoglobin expression and purification Support Protocol 1: Measuring O2 equilibration curves Support Protocol 2: Mass spectrometry to confirm NH2 -terminal acetylation.

Influence of cyano substituents on the electron density and catalytic activity towards the oxygen reduction reaction for iron phthalocyanine. The case for Fe(II) 2,3,9,10,16,17,23,24-octa(cyano)phthalocyanine

Iron(II) 2,3,9,10,16,17,23,24-octa(cyano)phthalocyanine (OCNFePc), was tested as a catalyst for the oxygen reduction reaction (ORR) adsorbed on carbon nanotubes. The composite was analyzed spectroscopically and electrochemically characterized at pH 13 and pH 1. The composite showed close to 4 electron processes at pH 13. Computational analysis indicates that the O2 molecule binds end-on to the metal center and that the dioxygen molecule is dissociated on both the Fe metal center and the corral ring. An Analysis of the molecular electrostatic potential confirms the behavior of cyano residues as electron-withdrawing moieties in OCNFePc. As a result of its catalytic behavior and theoretical analysis of its O2 binding energy, OCNFePc was placed in a high position on a volcano correlation of similar phthalocyanine composites.

Magnetic mechanism for the biological functioning of hemoglobin

The role of magnetism in the biological functioning of hemoglobin has been debated since its discovery by Pauling and Coryell in 1936. The hemoglobin molecule contains four heme groups each having a porphyrin layer with a Fe ion at the center. Here, we present combined density-functional theory and quantum Monte Carlo calculations for an effective model of Fe in a heme cluster. In comparison with these calculations, we analyze the experimental data on human adult hemoglobin (HbA) from the magnetic susceptibility, Mössbauer and magnetic circular dichroism (MCD) measurements. In both the deoxygenated (deoxy) and the oxygenated (oxy) cases, we show that local magnetic moments develop in the porphyrin layer with antiferromagnetic coupling to the Fe moment. Our calculations reproduce the magnetic susceptibility measurements on deoxy and oxy-HbA. For deoxy-HbA, we show that the anomalous MCD signal in the UV region is an experimental evidence for the presence of antiferromagnetic Fe-porphyrin correlations. The functional properties of hemoglobin such as the binding of O2, the Bohr effect and the cooperativity are explained based on the magnetic correlations. This analysis suggests that magnetism could be involved in the functioning of hemoglobin.

Targeted Myoglobin Delivery as a Strategy for Enhancing the Sensitivity of Hypoxic Cancer Cells to Radiation.

SummaryThe effectiveness of cancer radiotherapy is frequently hindered by the hypoxia of the tumor microenvironment. Direct delivery of oxygen to hypoxic tumor tissues is an attractive strategy to overcome this hypoxia-associated radioresistance. Herein, we report the generation of submicron-sized particles comprising myoglobin fused to the crystal-forming domain of Cry3Aa protein for the targeted delivery of oxygen to cancer cells. We demonstrate that myoglobin-containing particles were successfully produced in Bacillus thuringiensis with the assistance of the Cry3Aa domain I. Furthermore, these particles could be genetically modified to incorporate the cell penetrating peptide TAT and cell targeting peptide A549.1, resulting in particles that exhibited improved cellular uptake and targeting toward A549 cells. Notably, these myoglobin-containing particles increased the intracellular oxygen levels of A549 cells and thereby sensitized them to radiation. These findings suggest that the targeted delivery of O2-bound myoglobin could be an effective approach to enhance the efficacy of radiotherapy.