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Organohalide chemistry underpins many industrial and agricultural processes, and a large proportion of environmental pollutants are organohalides (1). Nevertheless, organohalide chemistry is not exclusively of anthropogenic origin, with natural abiotic and biological processes contributing to the global halide cycle (2, 3). Reductive dehalogenases are responsible for biological dehalogenation in organohalide respiring bacteria (4, 5), with substrates including polychlorinated biphenyls or dioxins (6, 7). Reductive dehalogenases form a distinct subfamily of cobalamin (B12)-dependent enzymes that are usually membrane associated and oxygen sensitive, hindering detailed studies (8, 12). Here we report the characterization of a soluble, oxygen-tolerant reductive dehalogenase and, by combining structure determination with EPR (electron paramagnetic resonance) spectroscopy and simulation, show that a direct interaction between the cobalamin cobalt and the substrate halogen underpins catalysis. In contrast to the carbon-cobalt bond chemistry catalysed by the other cobalamin-dependent subfamilies (13), we propose that reductive dehalogenases achieve reduction of the organohalide substrate via halogen-cobalt bond formation. This presents a new model in both organohalide and cobalamin (bio)chemistry that will guide future exploitation of these enzymes in bioremediation or biocatalysis., Cobalamin consists of a cobalt ion coordinated by a tetrapyrrole-derived macrocycle and readily forms a weak organometallic C-Co(III) bond (14). This bond can be homolytically or heterolytically cleaved and the [...]

Use of the pressure dependence of kinetic isotope effects, coupled with a study of their temperature dependence, as a probe for promoting motions in enzymatic hydrogen-tunneling reactions is reported. Employing morphinone reductase as our model system and by using stopped-flow methods, we measured the hydride transfer rate (a tunneling reaction) as a function of hydrostatic pressure and temperature. Increasing the pressure from 1 bar (1 bar = 100 kPa) to 2 kbar accelerates the hydride transfer reaction when both protium (from 50 to 161 [s.sup.-1] at 25[degrees]C) and deuterium (12 to 31 [s.sup.-1] at 25[degrees]C) are transferred. We found that the observed primary kinetic isotope effect increases with pressure (from 4.0 to 5.2 at 25[degrees]C), an observation incompatible with the Bell correction model for hydrogen tunneling but consistent with a full tunneling model. By numerical modeling, we show that both the pressure and temperature dependencies of the reaction rates are consistent with the framework of the environmentally coupled tunneling model of Kuznetsov and Ulstrup [Kuznetsov AM, Ulstrup J (1999) Can J Chem 77:1085-1096], providing additional support for the role of a promoting motion in the hydride tunneling reaction in morphinone reductase. Our study demonstrates the utility of 'barrier engineering' by using hydrostatic pressure as a probe for tunneling regimes in enzyme systems and provides added and independent support for the requirement of promoting motions in such tunneling reactions. flavoprotein | hydrogen tunneling | morphinone reductase | pressure dependence | stopped flow

Author(s): Mary Ortmayer [1]; Pierre Lafite [1]; Binuraj R. K. Menon [1]; Tewes Tralau [1]; Karl Fisher [1]; Lukas Denkhaus [1]; Nigel S. Scrutton [1]; Stephen E. J. Rigby [1]; [...]

[alpha]-Secondary kinetic isotope effects (2 degree KIEs) are used in conjunction with primary 1 degree KIE to investigate the mechanism of environmentally coupled hydrogen tunneling in the reductive half-reactions of two homologous flavoenzymes, morphine reductase and pentaerythritol tetranitrate reductase. The analysis has stressed on the benefit of combining studies of 1 degree and 2 degree KIEs to describe the pre-organization and local geometries within the context of contemporary environmentally coupled frameworks for H-tunneling.

The central photochemical reaction in photosystem II of green algae and plants and the reaction center of some photosynthetic bacteria involves a one-electron transfer from a light-activated chlorin complex to a bound quinone molecule. Through protein engineering, we have been able to modify a protein to mimic this reaction. A unique quinone-binding site was engineered into the Escherichia coil cytochrome [b.sub.562] by introducing a cysteine within the hydrophobic interior of the protein. Various quinones, such as p-benzoquinone and 2,3-dimethoxy-5-methyl-1,4-benzoquinone, were then covalently attached to the protein through a cysteine sulfur addition reaction to the quinone ring. The cysteine placement was designed to bind the quinone [approximately equal to] 10 [Angstrom] from the edge of the bound porphyrin. Fluorescence measurements confirmed that the bound hydroquinone is incorporated toward the protein's hydrophobic interior and is partially solvent-shielded. The bound quinones remain redox-active and can be oxidized and rereduced in a two-electron process at neutral pH. The semiquinone can be generated at high pH by a one-electron reduction, and the midpoint potential of this can be adjusted by [approximately equal to]500 mV by binding different quinones to the protein. The heme-binding site of the modified cytochrome was then reconstituted with the chlorophyll analogue zinc chlorin [e.sub.6]. By using EPR and fast optical techniques, we show that, in the various chlorin-protein-quinone complexes, light-induced electron transfer can occur from the chlorin to the bound oxidized quinone but not the hydroquinone, with electron transfer rates in the order of 10 (8) [s.sup.-1]. photosynthetic reaction center | artificial photosynthesis | chlorophyll analog | zinc chlorin | cysteine

Author(s): Karl A. P. Payne [1]; Mark D. White [1]; Karl Fisher [1]; Basile Khara [1]; Samuel S. Bailey [1]; David Parker [2]; Nicholas J. W. Rattray [1]; Drupad K. [...]

Author(s): Mark D. White [1]; Karl A. P. Payne [1]; Karl Fisher [1]; Stephen A. Marshall [1]; David Parker [2]; Nicholas J. W. Rattray [1]; Drupad K. Trivedi [1]; Royston [...]

The various effects of the donor-acceptor distance and the isotope effects on the force constant exhibited during barrier compression in the H-tunneling reactions are analyzed. The force constant is shown to highly increase with a decrease in the donor-acceptor relationship of the system.

The single hydride transfer from both NADPH and NADH to FMN in the reductive half-reaction of pentaerythritol tetranitrate reductase (PETNR) is examined. The studies have ruled out differences in rate due to variation in driving force and the donor-acceptor distance, which has shown that it is the promoting vibration with NADPH that is the origin of the increased observed rate.

A theoretical treatment of the combined effect of temperature and pressure on environmentally coupled H tunneling reactions is described. The pressure dependence of the kinetic isotope effect (KIE) for an H-transfer reaction has provided a method of determining the contribution of nuclear quantum mechanical (NQM) effects to the reaction, with a pressure-dependent KIE suggestive of NQM tunneling reaction.

The article shows that when it is rate-limiting in presteady-state kinetic studies with ethanolamine ammonia lyase (EAL), the net forward rate of C-Co bond homolysis is insensitive to the application of external magnetic fields in the presence of substrate. The studies have strong implications for the mechanism of radical pair (RP) stabilization and the unprecedented catalytic power of this class of cobalamin-dependent enzymes.

Stopped-flow spectroscopy has demonstrated that reduction of the FMN cofactor by NADH in N189A morphinone reductase (MR) in multiphasic, thus identifying four different reactive configurations of the MR-NADH complex. The analysis has helped in deriving the mechanistic information regarding multiple reactive configurations of the same enzyme-coenzyme complex by using stopped-flow methods.

Multiple solution-state methods are used to investigate the nature and control of electron transfer in the context of the proposed 'domain shuffle hypothesis' for intraprotein electron transfer inferred from the crystal structure of the nitric oxide synthase reductase domain. Solution data is provided that are consistent with the proposed domain shuffle hypothesis for electron transfer neuronal nitric oxide synthase (nNOS), inferred from the crystallographic structure of nNOS reductase.

The differences in the redox properties of protein-ligated 2,6-dimethylbenzoquinone (DMBQ) relative to the solvated quinine species are described. DMBQ, DMBQ thioether conjugated with N-acetyl-L-csysteine methyl) ester (bCys), and DMBQ thioether conjugated to [[alpha].sub.3]C are characterized by cyclic voltammetry using a 3-mercaptopropionoic acid modified gold electrode.

The differences in the redox properties of protein-ligated 2,6-dimethylbenzoquinone (DMBQ) relative to the solvated quinine species are described. DMBQ, DMBQ thioether conjugated with N-acetyl-L-csysteine methyl) ester (bCys), and DMBQ thioether conjugated to [[alpha].sub.3]C are characterized by cyclic voltammetry using a 3-mercaptopropionoic acid modified gold electrode.

The molecular dynamics simulations and density functional theory (DFT) calculations are applied to demonstrate that during proton tunneling in the oxidative deamination of tryptamine catalyzed by the enzyme aromatic amine dehydrogenase (AADH), a sub-picosecond promoting vibration is inherent to the iminoquinone intermediate. It is shown by numerical modeling that this short-range vibration is consistent with the gating motion in the hydrogen tunneling model in an enzymatic reaction with an observed protium/deuterium kinetic isotope effect that is not temperature-dependent.

De novo protein design and electrochemistry is used to measure changes in the potential and pK(sub A) of a phenol when its OH group is moved from a solvent-exposed to a sequestered protein position. A 'protein rotation strategy' was adopted in which phenols, containing a SH in position 4,3, or 2 relative to the OH group, were bound to a buried protein site.

The study describes the mutation of the iron-ligating methionine (Met) to a histidine (His), forming a protein capable of bis-His ligation of the heme iron. This protein is a double mutant that also has the His 63 to asparagine (H63N) mutation, which removes a solvent-exposed His.