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This paper is the first report of a P450-electrode in a microfluidic format. A 30 [micro]L microfluidic cell was made in poly(methyl methacrylate) containing the inlet, outlet, and reaction chamber with two electrode strips, one of which contains the human cytochrome P450 3A4 covalently bound to gold via a 6-bexanethiol and 7-mercaptoheptanoic acid (1:1) sell-assembled monolayer. The electrochemical response of the P450-electrode in the microfluidic cell was tested using four drags that are known substrates of P450 3A4: quinidine, nifedipine, alosetron and ondansetron. Titration experiments allowed the electrochemical measurements of KM for the four drugs, with values of 2.9, 29.1, 113.4, and 114.1 mM, respectively. The [K.sub.M] values are found to be in good agreement and correctly ranked with respect to the published literature on human liver microsomes and baculosomes: [ondansetron [approximately equal to] alosetron > nifedipine > quinidine]. The results presented in this paper represent a step forward for a rapid evaluation of the interaction of P450 and drug, requiring small volumes of new chemical entities to be tested. 10.1021/ac102480k

Oriented immobilization of human cytochrome P450 2E1 and its catalytic activity by direct electrochemistry was achieved by engineering two multisite mutants of P450 2E1: MUT261 (C268S-C480S-C488S) and MUT268 (C261S-C480S-C488S). Here, all the exposed cysteines are mutated into serines, with the exception of one (C261 for MUT261 and C268 for MUT268) that is able to link covalently to a modified gold electrode. The P450 2E1 wild type, as well as the two mutants, were immobilized onto gold electrodes using dithio-bismaleimidoethane as a self-assembled monolayer. The catalytic activity of the wild type and of the two cysteine mutants were determined using p-nitrophenol as a substrate, and the amount of the electrocatalysis product (p-nitrocatechol) was determined spectrophotometrically. The amounts of product formed by the mutants on the electrodes were 2-fold to 3-fold higher than those of the wild type. Control experiments performed in solution using the cytochrome P450 reductase as the electron donor show no significant differences in the level of product formed. The higher level of product formation of the two mutants on the electrode is ascribed to the controlled immobilization on the gold surface: the heine electron transfer proximal side is linked to the electrode, while the substrate binding distal side is exposed to the bulk solution. This is the first evidence that the control ever the orientation of the human cytochromes P450 is key to maximize the electrocatalytic efficiency of these enzymes. 10.1021/ac101072h

Site-directed mutagenesis and functionalization of gold surfaces have been combined to obtain a stable immobilization of the heme domain of cytochrome P450 BM3 from Bacillus megaterium. Immobilization experiments were carried out using the wild type protein bearing the surface C62 and C156 and the site-directed mutants C62S, the C156S, and the double mutant C62S/C156S (no exposed cysteines). The gold surface was functionalized using two different spacers: cystamine-N-succinimidyl 3-maleimidopropionate and dithio-bismaleimidoethane, both leading to the formation of maleimide-terminated monolayers capable of covalent linkage to cysteine. Tapping mode atomic force microscopy experiments carded out on cystamine-N-succinimidyl 3-male-imidopropionate derivatized gold led to good images with expected molecular heights (5.5-6.0 nm) for the wild type and the C156S mutant. These samples also gave measurable electrochemical signals with midpoint potentials of -48 and -58 mV for the wild type and C156S, respectively. On the other hand, the dithio-bismaleimidoethane spacer led to variability on the molecular heights measured by tapping mode atomic force microscopy and the electrochemical response. This is interpreted in terms of lack of homogeneous dithio-bismaleimidoethane monolayer on gold. Furthermore, results from tapping mode atomic force microscopy show that the double mutant and the C62S did not lead to stably immobilized P450 protein, confirming the necessity of the solvent exposed C62.

A spectroscopy chemical investigation of proton-coupled electron transfer is flavodoxin D- vulgaris hildenborough (Fld) is reported. The slow deprotonation step has a benefit to prevent undesirable loss of reducing the equivalents resulting from semiquinone oxidation to quinone.

This article discusses construction and functional characterization of two cytochrome c-553 homodimers from Desulfovibrio vulgaris to raise the reduction potential. The reduction potential increase is correlated with the water exclusion from the heme surface.

Photosystem II (PSII), the water/plastoquinone photo-oxidoreductase, plays a key energy input role in the biosphere. QA•-, the reduced semiquinone form of the nonexchangeable quinone, is often considered capable of a side reaction with O2, forming superoxide, but this reaction has not yet been demonstrated experimentally. Here, using chlorophyll fluorescence in plant PSII membranes, we show that O2 does oxidize QA•- at physiological O2 concentrations with a t1/2 of 10 s. Superoxide is formed stoichiometrically, and the reaction kinetics are controlled by the accessibility of O2 to a binding site near QA•-, with an apparent dissociation constant of 70 ± 20 µM. Unexpectedly, QA•- could only reduce O2 when bicarbonate was absent from its binding site on the nonheme iron (Fe2+) and the addition of bicarbonate or formate blocked the O2-dependant decay of QA•-. These results, together with molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics calculations, indicate that electron transfer from QA•- to O2 occurs when the O2 is bound to the empty bicarbonate site on Fe2+. A protective role for bicarbonate in PSII was recently reported, involving long-lived QA•- triggering bicarbonate dissociation from Fe2+ [Brinkert et al., Proc. Natl. Acad. Sci. U.S.A. 113, 12144-12149 (2016)]. The present findings extend this mechanism by showing that bicarbonate release allows O2 to bind to Fe2+ and to oxidize QA•-. This could be beneficial by oxidizing QA•- and by producing superoxide, a chemical signal for the overreduced state of the electron transfer chain. [ABSTRACT FROM AUTHOR]

The electrochemical study of the immobilized human P450 2E1 either absorbed or covalently linked to different electrode surfaces is presented. The findings indicate that the immobilized enzyme responds to the presence of the substrate in a Michaelis- Menten fashion.

Photosystem II (PSII), the light-driven water/plastoquinone photooxidoreductase, is of central importance in the planetary energy cycle. The product of the reaction, plastohydroquinone (PQH2), is released into the membrane from the QB site, where it is formed. A plastoquinone (PQ) from the membrane pool then binds into the QB site. Despite their functional importance, the thermodynamic properties of the PQ in the QB site, QB, in its different redox forms have received relatively little attention. Here we report the midpoint potentials (Em) of QB in PSII from Thermosynechococcus elongatus using electron paramagnetic resonance (EPR) spectroscopy: Em QB/QB •- ≈ 90 mV, and Em QB •-/QBH2 ≈ 40 mV. These data allow the following conclusions: 1) The semiquinone, QB •-, is stabilized thermodynamically; 2) the resulting Em QB/QBH2 (~65 mV) is lower than the Em PQ/PQH2 (~117 mV), and the difference (ΔE ≈ 50 meV) represents the driving force for QBH2 release into the pool; 3) PQ is ~50× more tightly bound than PQH2; and 4) the difference between the Em QB/QB •- measured here and the Em QA/QA •- from the literature is ~234 meV, in principle corresponding to the driving force for electron transfer from QA •- to QB. The pH dependence of the thermoluminescence associated with QB •- provided a functional estimate for this energy gap and gave a similar value (≥180 meV). These estimates are larger than the generally accepted value (~70 meV), and this is discussed. The energetics of QB in PSII are comparable to those in the homologous purple bacterial reaction center. [ABSTRACT FROM AUTHOR]