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In thylakoid membranes, photosystem II (PSII) monomers from the stromal lamellae contain the subunits PsbS and Psb27 (PSIIm-S/27), while PSII monomers (PSIIm) from granal regions lack these subunits. Here, we have isolated and characterized these 2 types of PSII complexes in tobacco (Nicotiana tabacum). PSIIm-S/27 showed enhanced fluorescence, the near absence of oxygen evolution, and limited and slow electron transfer from QA to QB compared to the near-normal activities in the granal PSIIm. However, when bicarbonate was added to PSIIm-S/27, water splitting and QA to QB electron transfer rates were comparable to those in granal PSIIm. The findings suggest that the binding of PsbS and/or Psb27 inhibits forward electron transfer and lowers the binding affinity for bicarbonate. This can be rationalized in terms of the recently discovered photoprotection role played by bicarbonate binding via the redox tuning of the QA/QA•− couple, which controls the charge recombination route, and this limits chlorophyll triplet-mediated 1O2 formation. These findings suggest that PSIIm-S/27 is an intermediate in the assembly of PSII in which PsbS and/or Psb27 restrict PSII activity while in transit using a bicarbonate-mediated switch and protective mechanism.

The Mo/Fe nitrogenase enzyme is unique in its ability to efficiently reduce dinitrogen to ammonia at atmospheric pressures and room temperature. Should an artificial electrolytic device achieve the same feat, it would revolutionise fertilizers and even provide an energy dense, truly carbon-free fuel. This Review provides a coherent comparison of recent progress made in dinitrogen fixation on (i) solid electrodes, (ii) homogeneous catalysts and (iii) nitrogenases. Specific emphasis is placed on systems for which there is unequivocal evidence that dinitrogen reduction has taken place. By establishing the cross-cutting themes and synergies between these systems, we identify viable avenues for future research.

In thylakoid membranes, Photosystem II monomers from the stromal lamellae contain the subunits PsbS and Psb27 (PSIIm-S/27), while Photosystem II monomers from granal regions (PSIIm) lack these subunits. Here, we have isolated and characterised these two types of Photosystem II complexes. The PSIIm-S/27 showed enhanced fluorescence, the near-absence of oxygen evolution, as well as limited and slow electron transfer from QA to QB compared to the near-normal activities in the granal PSIIm. However, when bicarbonate was added to the PSIIm-S/27, water splitting and QA to QB electron transfer rates were comparable to those in granal PSIIm. The findings suggest that the binding of PsbS and/or Psb27 inhibits forward electron transfer and lowers the binding affinity for the bicarbonate. This can be rationalized in terms of the recently discovered photoprotection role played by bicarbonate binding via the redox tuning of the QA/QA•− couple, which controls the charge recombination route, and this limits chlorophyll triplet mediated 1O2 formation (Brinkert K et al. (2016) Proc Natl Acad Sci U S A. 113(43):12144-12149). These findings suggest that PSIIm-S/27 is an intermediate in the assembly of PSII in which PsbS and/or Psb27 restrict PSII activity while in transit, by using a bicarbonate-mediated switch and protective mechanism.One sentence summaryA photosystem II monomer with PsbS and Psb27 as additional subunits, is inactive as isolated but activated by bicarbonate, and is attributed to be a late-stage intermediate in photoassembly.

Significance In Photosystem II (PSII), O 2 reduction by Q A • − is often discussed but has not been demonstrated. Here, we show in PSII membranes that Q A • − can reduce O 2 to superoxide, but only when bicarbonate is absent from its binding site on the nonheme Fe 2+ . Bicarbonate’s role in PSII was recently shown to involve a regulatory/protective redox-tuning mechanism linking PSII function to CO 2 concentration. A key aspect is the presence of stable Q A • − causing release of bicarbonate from its site on Fe 2+ . Here, we show that under these conditions, O 2 binds to the empty site on the Fe 2+ and is reduced by Q A • − . This unexpected reaction may be a further indication of cross-talk between the regulation of PSII and CO 2 fixation.

Photosystem II (PSII), the water/plastoquinone photo-oxidoreductase, plays a key energy input role in the biosphere. [Formula: see text], the reduced semiquinone form of the nonexchangeable quinone, is often considered capable of a side reaction with O

Significance The E m values of the 2 Q B redox couples, E m (Q B /Q B •− ) and E m (Q B •− /Q B H 2 ), in Photosystem II are fundamental for understanding the enzyme function, but they remain vague. Using EPR signals arising from the semiquinone, Q B •− , we have now measured both redox couples. The semiquinone is thermodynamically stable and has a relatively high potential. This minimizes back-reactions and electrons leaking onto O 2 , explaining the remarkable stability of Q B •− . The release of Q B H 2 is thermodynamically favorable, and the binding of the substrate PQ is greatly favored over the product PQH 2 : This optimizes PSII function in the presence of a largely reduced plastoquinone pool. This work corrects and explains a recent report of anomalous values for the Q B redox couples.

The recently discovered, chlorophyll-f-containing, far-red photosystem II (FR-PSII) supports far-red light photosynthesis. Participation and kinetics of spectrally shifted far-red pigments are directly observable and separated from that of bulk chlorophyll-a. We present an ultrafast transient absorption study of FR-PSII, investigating energy transfer and charge separation processes. Results show a rapid subpicosecond energy transfer from chlorophyll-a to the long-wavelength chlorophylls-f/d. The data demonstrate the decay of an ∼720-nm negative feature on the picosecond-to-nanosecond timescales, coinciding with charge separation, secondary electron transfer, and stimulated emission decay. An ∼675-nm bleach attributed to the loss of chl-a absorption due to the formation of a cation radical, PD1+•, is only fully developed in the nanosecond spectra, indicating an unusually delayed formation. A major spectral feature on the nanosecond timescale at 725 nm is attributed to an electrochromic blue shift of a FR-chlorophyll among the reaction center pigments. These time-resolved observations provide direct experimental support for the model of Nürnberg et al. [D. J. Nürnberg et al., Science 360, 1210–1213 (2018)], in which the primary electron donor is a FR-chlorophyll and the secondary donor is chlorophyll-a (PD1 of the central chlorophyll pair). Efficient charge separation also occurs using selective excitation of long-wavelength chlorophylls-f/d, and the localization of the excited state on P720* points to a smaller (entropic) energy loss compared to conventional PSII, where the excited state is shared over all of the chlorin pigments. This has important repercussions on understanding the overall energetics of excitation energy transfer and charge separation reactions in FR-PSII.

Far-red light (FRL) Photosystem II (PSII) isolated from Chroococcidiopsis thermalis is studied using parallel analyses of low-temperature absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies in conjunction with fluorescence measurements. This extends earlier studies (Nurnberg et al 2018 Science 360 (2018) 1210-1213). We confirm that the chlorophyll absorbing at 726 nm is the primary electron donor. At 1.8 K efficient photochemistry occurs when exciting at 726 nm and shorter wavelengths; but not at wavelengths longer than 726 nm. The 726 nm absorption peak exhibits a 21 ± 4 cm−1 electrochromic shift due to formation of the semiquinone anion, QA•-. Modelling indicates that no other FRL pigment is located among the 6 central reaction center chlorins: PD1, PD2 ChlD1, ChlD2, PheoD1 and PheoD2. Two of these chlorins, ChlD1 and PD2, are located at a distance and orientation relative to QA•- so as to account for the observed electrochromic shift. Previously, ChlD1 was taken as the most likely candidate for the primary donor based on spectroscopy, sequence analysis and mechanistic arguments. Here, a more detailed comparison of the spectroscopic data with exciton modelling of the electrochromic pattern indicates that PD2 is at least as likely as ChlD1 to be responsible for the 726 nm absorption. The correspondence in sign and magnitude of the CD observed at 726 nm with that predicted from modelling favors PD2 as the primary donor. The pros and cons of PD2 vs ChlD1 as the location of the FRL-primary donor are discussed.TOC GraphicHighlightsPrimary Donor confirmed at 726 nmDetermination of far-red chl pigment Qy excitation positions, widths, CD and MCD amplitudesQuantification of electrochromic shifts and QA•- photoconversion yieldElectrochromic shift consistent with primary donor at either ChlD1 or PD2The CD amplitude favors the primary donor at PD2

Far-red light (FRL) Photosystem II (PSII) isolated from Chroococcidiopsis thermalis is studied using parallel analyses of low-temperature absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies in conjunction with fluorescence measurements. This extends earlier studies (Nurnberg et al 2018 Science 360 (2018) 1210-1213). We confirm that the chlorophyll absorbing at 726 nm is the primary electron donor. At 1.8 K efficient photochemistry occurs when exciting at 726 nm and shorter wavelengths; but not at wavelengths longer than 726 nm. The 726 nm absorption peak exhibits a 21 ± 4 cm

Photosystem II (PSII), the light-driven water/plastoquinone photooxidoreductase, is of central importance in the planetary energy cycle. The product of the reaction, plastohydroquinone (PQH(2)), is released into the membrane from the Q(B) site, where it is formed. A plastoquinone (PQ) from the membrane pool then binds into the Q(B) site. Despite their functional importance, the thermodynamic properties of the PQ in the Q(B) site, Q(B), in its different redox forms have received relatively little attention. Here we report the midpoint potentials (E(m)) of Q(B) in PSII from Thermosynechococcus elongatus using electron paramagnetic resonance (EPR) spectroscopy: E(m) Q(B)/Q(B)(•−) ≈ 90 mV, and E(m) Q(B)(•−)/Q(B)H(2) ≈ 40 mV. These data allow the following conclusions: 1) The semiquinone, Q(B)(•−), is stabilized thermodynamically; 2) the resulting E(m) Q(B)/Q(B)H(2) (∼65 mV) is lower than the E(m) PQ/PQH(2) (∼117 mV), and the difference (ΔE ≈ 50 meV) represents the driving force for Q(B)H(2) release into the pool; 3) PQ is ∼50× more tightly bound than PQH(2); and 4) the difference between the E(m) Q(B)/Q(B)(•−) measured here and the E(m) Q(A)/Q(A)(•−) from the literature is ∼234 meV, in principle corresponding to the driving force for electron transfer from Q(A)(•−) to Q(B). The pH dependence of the thermoluminescence associated with Q(B)(•−) 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 Q(B) in PSII are comparable to those in the homologous purple bacterial reaction center.

The effect of bicarbonate (HCO3-) on photosystem II (PSII) activity was discovered in the 1950s, but only recently have its molecular mechanisms begun to be clarified. Two chemical mechanisms have been proposed. One is for the electron-donor side, in which mobile HCO3- enhances and possibly regulates water oxidation by acting as proton acceptor, after which it dissociates into CO2 and H2O. The other is for the electron-acceptor side, in which (i) reduction of the Q(A) quinone leads to the release of HCO(3)(- )from its binding site on the non-heme iron and (ii) the E-m potential of the Q(A)/Q(.-) couple increases when HCO3- dissociates. This suggested a protective/regulatory role of HCO3- as it is known that increasing the E-m of Q(A) decreases the extent of back-reaction-associated photodamage. Here we demonstrate, using plant thylakoids, that time-resolved membrane-inlet mass spectrometry together with C-13 isotope labeling of HCO3- allows donor- and acceptor-side formation of CO2 by PSII to be demonstrated and distinguished, which opens the door for future studies of the importance of both mechanisms under in vivo conditions.

Bicarbonate removal from the nonheme iron at the acceptor side of photosystem II (PSII) was shown recently to shift the midpoint potential of the primary quinone acceptor Q(A) to a more positive potential and lowers the yield of singlet oxygen ((1)O(2)) production. The presence of Q(A)(−) results in weaker binding of bicarbonate, suggesting a redox-based regulatory and protective mechanism where loss of bicarbonate or exchange of bicarbonate by other small carboxylic acids may protect PSII against (1)O(2) in vivo under photorespiratory conditions. Here, we compared the properties of Q(A) in the Arabidopsis (Arabidopsis thaliana) photorespiration mutant deficient in peroxisomal HYDROXYPYRUVATE REDUCTASE1 (hpr1-1), which accumulates glycolate in leaves, with the wild type. Photosynthetic electron transport was affected in the mutant, and chlorophyll fluorescence showed slower electron transport between Q(A) and Q(B) in the mutant. Glycolate induced an increase in the temperature maximum of thermoluminescence emission, indicating a shift of the midpoint potential of Q(A) to a more positive value. The yield of (1)O(2) production was lowered in thylakoid membranes isolated from hpr1-1 compared with the wild type, consistent with a higher potential of Q(A)/Q(A)(−). In addition, electron donation to photosystem I was affected in hpr1-1 at higher light intensities, consistent with diminished electron transfer out of PSII. This study indicates that replacement of bicarbonate at the nonheme iron by a small carboxylate anion occurs in plants in vivo. These findings suggested that replacement of the bicarbonate on the nonheme iron by glycolate may represent a regulatory mechanism that protects PSII against photooxidative stress under low-CO(2) conditions.

The biological route for nitrogen gas entering the biosphere is reduction to ammonia by the nitrogenase enzyme, which is inactivated by oxygen. Three types of nitrogenase exist, the least studied of which is the iron-only nitrogenase. The Anf3 protein in the bacterium Rhodobacter capsulatus is essential for diazotrophic (i.e. nitrogen-fixing) growth with the iron-only nitrogenase, but its enzymatic activity and function are unknown. Here, we biochemically and structurally characterize Anf3 from the model diazotrophic bacterium Azotobacter vinelandii. Determining the Anf3 crystal structure to atomic resolution, we observed that it is a dimeric flavocytochrome with an unusually close interaction between the heme and the flavin adenine dinucleotide cofactors. Measuring the reduction potentials by spectroelectrochemical redox titration, we observed values of -420 ± 10 mV and -330 ± 10 mV for the two FAD potentials and -340 ± 1 mV for the heme. We further show that Anf3 accepts electrons from spinach ferredoxin and that Anf3 consumes oxygen without generating superoxide or hydrogen peroxide. We predict that Anf3 protects the iron-only nitrogenase from oxygen inactivation by functioning as an oxidase in respiratory protection, with flavodoxin or ferredoxin as the physiological electron donors.

The biological route for nitrogen gas entering the biosphere is reduction to ammonia by the nitrogenase enzyme, which is inactivated by oxygen. Three types of nitrogenase exist, the least-studied of which is the iron-only nitrogenase. The Anf3 protein in the bacterium Rhodobacter capsulatus is essential for diazotrophic (i.e. nitrogen-fixing) growth with the iron-only nitrogenase, but its enzymatic activity and function are unknown. Here, we biochemically and structurally characterize Anf3 from the model diazotrophic bacterium Azotobacter vinelandii. Determining the Anf3 crystal structure to atomic resolution, we observed that it is a dimeric flavocytochrome with an unusually close interaction between the heme and the FAD cofactors. Measuring the reduction potentials by spectroelectrochemical redox titration, we observed values of −420 ± 10 and −330 ± 10 mV for the two FAD potentials and −340 ± 1 mV for the heme. We further show that Anf3 accepts electrons from spinach ferredoxin and that Anf3 consumes oxygen without generating superoxide or hydrogen peroxide. We predict that Anf3 protects the iron-only nitrogenase from oxygen inactivation by functioning as an oxidase in respiratory protection, with flavodoxin or ferredoxin as the physiological electron donors.

Protein film photoelectrochemistry has previously been used to monitor the activity of photosystem II, the water-plastoquinone photooxidoreductase, but the mechanistic information attainable from a three-electrode setup has remained limited. Here we introduce the four-electrode rotating ring disk electrode technique for quantifying light-driven reaction kinetics and mechanistic pathways in real time at the enzyme–electrode interface. This setup allows us to study photochemical H2O oxidation in photosystem II and to gain an in-depth understanding of pathways that generate reactive oxygen species. The results show that photosystem II reacts with O2 through two main pathways that both involve a superoxide intermediate to produce H2O2. The first pathway involves the established chlorophyll triplet-mediated formation of singlet oxygen, which is followed by its reduction to superoxide at the electrode surface. The second pathway is specific for the enzyme/electrode interface: an exposed antenna chlorophyll is sufficiently close to the electrode for rapid injection of an electron to form a highly reducing chlorophyll anion, which reacts with O2 in solution to produce O2•–. Incomplete H2O oxidation does not significantly contribute to reactive oxygen formation in our conditions. The rotating ring disk electrode technique allows the chemical reactivity of photosystem II to be studied electrochemically and opens several avenues for future investigation.

Factors governing the photoelectrochemical output of photosynthetic microorganisms are poorly understood, and energy loss may occur due to inefficient electron transfer (ET) processes. Here, we systematically compare the photoelectrochemistry of photosystem II (PSII) protein-films to cyanobacteria biofilms to derive: (i) the losses in light-to-charge conversion efficiencies, (ii) gains in photocatalytic longevity, and (iii) insights into the ET mechanism at the biofilm interface. This study was enabled by the use of hierarchically structured electrodes, which could be tailored for high/stable loadings of PSII core complexes and Synechocystis sp. PCC 6803 cells. The mediated photocurrent densities generated by the biofilm were 2 orders of magnitude lower than those of the protein-film. This was partly attributed to a lower photocatalyst loading as the rate of mediated electron extraction from PSII in vitro is only double that of PSII in vivo. On the other hand, the biofilm exhibited much greater longevity (>5 days) than the protein-film (

Significance The E m of Q A / Q A − • is the reference pegging the thermodynamics of Photosystem II (PSII) to an absolute value. This work resolves a long-standing discrepancy in the literature, removing this ambiguity at the heart of PSII bioenergetics. The discrepancy in the literature reflects the loss of bicarbonate in some titrations. This surprising finding shows that bicarbonate binding controls the E m of Q A / Q A − • , and thus the redox state of Q A influences the binding of bicarbonate. This process constitutes a previously unrecognized regulation of PSII: Q A − • triggers bicarbonate release, slowing electron transfer and tuning the potential of Q A to protect PSII against photodamage. This work provides an explanation for the existence of bicarbonate in PSII, a mystery for more than half a century.

Over the past two decades, a wealth of information on the human cytochrome P450 enzymes and their role in drug metabolism both in vitro and in vivo has been gathered. Our understanding of this area has progressed greatly, but our confidence in the development of quantitative projections of drug interactions, made from in vitro data, is somehow still shaky. There are therefore no doubts in the necessity for reliable and fast methodologies for P450 drug metabolism analysis, capable of providing accurate and precise in vitro data. This paper reports on the first integration of a P450-electrode into a microtiter plate format for the rapid determination of the affinity parameters (K(M)) for a set of known drugs. The most relevant human drug metabolizing cytochromes P450, isoforms 3A4, 2D6, and 2C9, have been covalently bound to a gold electrode via a 10-carboxydecanethiol and 8-hydroxyoctanethiol (1:1) self-assembled monolayer at the bottom of an eight-well microtiter plate. The electrochemical response of the P450-electrode and the performance of the platform have been validated using a set of 30 known drugs with K(M) values spanning from less than 1 to more than 100 μM. The K(M) values obtained using this platform show an excellent error, and their ranking is within the range of those present in the literature determined from conventional incubation experiments with cytochrome P450s 3A4, 2D6, and 2C9.

Improving the electrochemical performance of cytochrome P450 enzymes is highly desirable due to their versatility in the recognition of different biological and xenobiotic compounds. The task poses an exciting challenge because it leads not only to the acquisition of fundamental knowledge on the redox properties of these enzymes, but it also opens opportunities for technological and commercial applications. Interfacing these enzymes to electrode surfaces and electrochemically driving their catalytic cycle has proven to be very difficult. Initial attempts made by several groups included the direct immobilisation of these enzymes on electrode surfaces and omission of their redox partners for simplification of their electron transfer pathway. The data obtained in these cases generally resulted in a high heterogeneous electron transfer rate but without success in terms of detectable substrate turnover. The breakthrough in electrocatalysis has been made when both the electrode and the enzyme have been engineered, in some cases mimicking the natural environment of the microsomal enzymes and the inclusion of their electron transfer partners. This paper reviews and discusses the recent literature on this subject, and highlights the different approaches that have led to an unprecedented advancement of this area of research.

This paper is the first report of a P450-electrode in a microfluidic format. A 30 μL 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-hexanethiol and 7-mercaptoheptanoic acid (1:1) self-assembled monolayer. The electrochemical response of the P450-electrode in the microfluidic cell was tested using four drugs that are known substrates of P450 3A4: quinidine, nifedipine, alosetron and ondansetron. Titration experiments allowed the electrochemical measurements of K(M) for the four drugs, with values of 2.9, 29.1, 113.4, and 114.1 mM, respectively. The K(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 ≈ alosetronnifedipinequinidine]. 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.

THE WORLD ENERGY PROBLEM AND THE BIOFUEL ENERGY PROBLEM Oxygenic photosynthesis has been promoted as a system for fuel production on a global scale to replace fossil fuels. The fundamental requirement for this to be viable is that the energy output of the system must be greater than the energy input from fossil fuels. For biofuel production, this criterion is not always met. This issue is often dodged because life-cycle analyses are complex (and thus disputed) and future technological innovations can always be invoked. The second requirement is a sufficiently high rate of solar energy conversion to make the process feasible in terms of the time and space needed to produce fuel on a relevant scale. Both requirements are closely linked to the photosynthetic efficiency; i.e., the conversion efficiency of solar energy to organic material (sugar, biomass, hydrocarbons, etc.). Here, we discuss limitations on photosynthetic efficiency and approaches suggested to overcome them. We focus on biofuels produced by photosynthetic microbes as they are often considered the fuels of the future for their year-round cultivation, non-competition with food crops, higher reported photosynthetic yields, and the potential for genetic engineering to produce fuels directly (Brennan and Owende, 2010). The energy investment required for biomass production (e.g., water, nutrients, fertilizers, stirring, bubbling, containment, harvesting, processing) cancels out some or all of the energy gained from sunlight (Slade and Bauen, 2013). This is described by the energy returned as a proportion of energy invested (EROI), and this factor is the key measure of energy sustainability in life-cycle analysis (Murphy and Hall, 2010). If the EROI is >1, the system produces a fuel with net solar energy content; if 1 and a high rate of solar energy conversion) on a pilot scale seems advisable before scaling-up is considered. The energetic prerequisites rely fundamentally on the efficiency of photosynthesis. Calculations for theoretical photosynthetic efficiency agree on a maximum value for solar energy to carbon–carbon bonds in glucose of around 13%, falling to around 5% of solar energy to biomass for C3 plants, considering photorespiratory and respiratory losses (Zhu et al., 2010). The highest efficiency reported for photosynthetic microbes under controlled lab conditions is 3% for light-to-biomass [Melis, 2009; also Cuaresma et al. (2009)]. Under growth conditions more relevant to industrial settings, the efficiency is stated to be significantly lower than this (Melis, 2009). Efforts are thus being made to find ways of improving photosynthesis itself.

Cellular Retinol-Binding Protein type I (CRBP) exhibits very high affinity for its ligand, bound within a buried cavity completely shielded from the outside medium. Three-dimensional structure and backbone dynamics in aqueous solution at neutral pH, either in the absence or in the presence of retinol, fail to represent the protein in a state capable of ligand uptake and release. The question was asked whether changes in the composition of the outside medium might facilitate ligand dissociation. Acidic aqueous solutions and water-alcohol mixtures were selected, among the best described denaturing solvents, to investigate their effects on the stability of the carrier-ligand complex and the conformational state of the protein upon ligand release. Circular dichroism (CD) and fluorescence spectroscopy were used to probe protein secondary and tertiary structure, compactness and retinol dissociation. While in purely aqueous media retinol dissociation parallels the acid-induced denaturation of the carrier, in water-alcohol mixtures it occurs in a range of co-solvent content lower than that required for protein denaturation. In light of these results, it is suggested that local solvent properties in vivo might modulate protein conformation and flexibility and thus play a fundamental role in the control of retinol exchange between carrier and membrane-bound donors and acceptors.

In this paper, we report a spectroelectrochemical investigation of proton-coupled electron transfer in flavodoxin D. vulgaris Hildenborough (Fld). Poly-L-lysine is used to promote the binding of Fld to the nanocrystalline, mesoporous SnO(2) electrodes. Two reversible redox couples of the immobilized Fld are observed electrochemically and are assigned by spectroelectrochemistry to the quinone/semiquinone and semiquinone/hydroquinone couples of the protein's flavin mononucleotide (FMN) redox cofactor. Comparison with control data for free FMN indicates no contamination of the Fld data by dissociated FMN. The quinone/semiquinone and semiquinone/hydroquinone midpoint potentials (E(q/sq) and E(sq/hq)) at pH 7 were determined to be -340 and -585 mV vs Ag/AgCl, in good agreement with the literature. E(q/sq) exhibited a pH dependence of 51 mV/pH. The kinetics of these redox couples were studied using cyclic voltammetry, cyclic voltabsorptometry, and chronoabsorptometry. The semiquinone/quinone reoxidation is found to exhibit slow, potential-independent but pH-sensitive kinetics with a reoxidation rate constant varying from 1.56 s(-)(1) at pH 10 to 0.0074 s(-)(1) at pH 5. The slow kinetics are discussed in terms of a simple kinetics model and are assigned to the reoxidation process being rate limited by semiquinone deprotonation. It is proposed that this slow deprotonation step has the physiological benefit of preventing the undesirable loss of reducing equivalents which results from semiquinone oxidation to quinone.

Engineered metalloproteins offer interesting systems for electrochemical studies of protein structure/function and their applications in nanobiotechnology. Scanning probe microscopy and cyclic voltammetry of engineered metalloproteins and electrodes have proved to be a powerful combination of tools contributing to the field of bioelectrochemistry. The ability to engineer tags, such as histidine tags and biotin-acceptor peptides, and to site-specifically introduce cysteine residues enabled the creation of ordered immobilised protein structures that can be characterised both electrochemically and topographically. Gene fusion and de novo combinatorial synthesis of metalloproteins are emerging to provide structures with the desired electrochemical properties.

This is the first report of the direct electrochemistry of the reductase (PHR) and oxygenase (PHO) components of phenol hydroxylase from Acinetobacter radioresistens S13 studied by cyclic and differential pulse voltammetry. The PHR contains one 2Fe2S cluster and one FAD that mediate the transfer of electrons from NAD(P)H to the non-heme diiron cluster of PHO. Cyclic and differential pulse voltammetry (CV and DPV) on glassy carbon showed two redox pairs with midpoint potentials at +131.5 ± 13 mV and -234 ± 3 mV versus normal hydrogen electrode (NHE). The first redox couple is attributed to the FeS centre, while the second one corresponds to free FAD released by the protein. DPV scans on native and guanidinium chloride treated PHR highlighted the presence of a split signal (ΔE ≈ 100 mV) attributed to heterogeneous properties of the 2Fe2S cluster interacting with the electrode, possibly due to the presence of two protein conformers and consistently with the large peak-to-peak separation and the peak broadening observed in CV. DPV experiments on gold electrodes performed on PHO confirm a consistently higher reduction potential at +396 mV vs. NHE. The positive redox potentials measured by direct electrochemistry for the FeS cluster in PHR and for the non-heme diiron cluster of PHO show that the entire phenol hydroxylase system works at higher potentials than those reported for structurally similar enzymes, for example methane monooxygenases.

"Personalized medicine" is a new concept in health care, one aspect of which defines the specificity and dosage of drugs according to effectiveness and safety for each patient. Dosage strongly depends from the rate of metabolism which is primarily regulated by the activity of cytochrome P450. In addition to the need for a genetic characterization of the patients, there is also the necessity to determine the drug-clearance properties of the polymorphic P450 enzyme. To address this issue, human P450 2D6 and 2C9 were engineered and covalently linked to an electrode surface allowing fast, accurate, and reliable measurements of the kinetic parameters of these phase-1 drug metabolizing polymorphic enzymes. In particular, the catalytic activity of P450 2C9 on the electrode surface was found to be improved when expressed from a gene-fusion with flavodoxin from Desulfovibrio vulgaris (2C9/FLD). The results are validated using marker drugs for these enzymes, bufuralol for 2D6, and warfarin for 2C9/FLD. The platform is able to measure the same small differences in K(M), and it allows a fast and reproducible mean to generated the product identified by HPLC from which the k(cat) is calculated.

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 heme 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 over the orientation of the human cytochromes P450 is key to maximize the electrocatalytic efficiency of these enzymes.

The mutant S64C of the short-chain flavodoxin from Desulfovibrio vulgaris has been designed to introduce an accessible and reactive group on the protein surface. Crystals have been obtained of both the monomeric and homodimeric forms of the protein, with the cofactor FMN in either the oxidized or the one electron-reduced (semiquinone) state, and the structures have been determined to high resolution. The redox properties of the different species have been investigated and the variations observed with respect to wild type have been related to the structural changes induced by the mutation and S–S bridge formation.

Investigation of the complex formation and electron transfer kinetics between P450 BMP and flavodoxin was carried out following the suggested involvement of flavodoxin in modulating the electron transfer to BMP in artificial redox chains bound to an electrode surface. While electron transfer measurements show the formation of a tightly bound complex, the NMR data indicate the formation of shortly lived complexes. The measured k obs ranged from 24.2 s − 1 to 44.1 s − 1 with k on ranging from 0.07 × 10 6 to 1.1 × 10 6 s − 1 M − 1 and K d ranging from 300 μM to 24 μM in buffers of different ionic strength. This apparent contradiction is due to the existence of two events in the complex formation prior to electron transfer. A stable complex is initially formed. Within such tightly bound complex, flavodoxin rocks rapidly between different positions. The rocking of the bound flavodoxin between several different orientations gives rise to the transient complexes in fast exchange as observed in the NMR experiments. Docking simulations with two different approaches support the theory that there is no highly specific orientation in the complex, but instead one side of the flavodoxin binds the P450 with high overall affinity but with a number of different orientations. The level of functionality of each orientation is dependent on the distance between cofactors, which can vary between 8 and 25 A, with some of the transient complexes showing distances compatible with the measured electron transfer rate constants.

This study shows that regulating the electron flow to the heme of human cytochrome P450 CYP3A4, using artificial redox chains, can significantly enhance its coupling efficiency and catalytic activity at electrode surfaces. The human CYP3A4 was fused at the genetic level either to the reductase domain of CYP102A1 (BMR) to create the CYP3A4/BMR or to Desulfovibrio vulgaris flavodoxin (FLD) to create the CYP3A4/FLD. Direct electrochemistry of the CYP3A4, CYP3A4/BMR and CYP3A4/FLD on glassy carbon and gold electrodes showed that the BMR and FLD flavo-proteins reduced the electron transfer rate to the CYP3A4 heme. Electrocatalysis resulted in appreciably higher product formation with the immobilized CYP3A4/BMR and CYP3A4/FLD on both surfaces due to an increased coupling efficiency. Rotating disk electrode studies and quantification of hydrogen peroxide were consistent with the proposed mechanism of a longer lived iron-peroxy species in the immobilized CYP3A4/BMR and CYP3A4/FLD. The approaches in this study provide a better understanding of cytochrome P450 uncoupling at electrode surfaces and aids in the construction of improved cytochrome P450 biosensors and bioelectrocatalysts. Keywords: Bioelectrochemistry, Protein engineering, Cytochrome P450, Electron transfer, Uncoupling

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 carried out on cystamine- N-succinimidyl 3-maleimidopropionate 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.

Cyclic voltabsorptometry is used for the first time to distinguish and characterize electrochemically the active (P450) and inactive (P420) forms of cytochromes P450 immobilized on an electrode during voltammetry experiments. This was achieved by using the heme domain (BMP) of the bacterial cytochrome P450 BM3 from Bacillus megaterium (CYP102A1) immobilized on mesopouros tin-oxide (SnO2) electrodes. We demonstrate that the formation of either the P450 form or the P420 one can be obtained by modifying the mesoporous electrode surface with polycations with different properties such as polyethylenimmine (PEI) and polydiallyldimethylammonium chloride (PDDA). Potential step spectroelectrochemistry allowed measurement of reduction potentials of the active P450 form. Values of -0.39+/-0.01 V and -0.58+/-0.01 V (both versus Ag/AgCl) were calculated for the active P450 form immobilized on the BMP/PDDA-SnO2 and BMP/PEI-SnO2 electrodes, respectively. The cyclic voltabsorptometric experiments showed how, when both the active and inactive forms are present on the PEI film, the inactive P420 species tends to dominate the cyclic voltammetric signal.

This work provides functional data showing that the bacterial CYP102A1 recognises compounds metabolised by human CYP3A4, CYP2E1 and CYP1A2 and is able to catalyse different reactions. Wild-type cytochrome CYP102A1 from Bacillus megaterium is a catalytically self-sufficient enzyme, containing an NADPH-dependent reductase and a P450 haem domain fused in a single polypeptidie chain. An NADPH-dependent method (Tsotsou et al. in Biosens. Bioelectron. 17:119–131, 2002) together with spectroscopic assays were applied to investigate the catalytic activity of CYP102A1 towards 19 xenobiotics, including 17 commercial drugs. These molecules were chosen to represent typical substrates of the five main families of drug-metabolising human cytochromes P450. Liquid chromatography–mass spectrometry analysis showed that CYP102A1 catalyses the hydroxylation of chlorzoxazone, aniline and p-nitrophenol, as well as the N-dealkylation of propranolol and the dehydrogenation of nifedipine. These drugs are typical substrates of human CYP2E1 and CYP3A4. The K M values calculated for these compounds were in the millimolar range: 1.21 ± 0.07 mM for chlorzoxazone, 2.52 ± 0.08 mM for aniline, 0.81 ± 0.04 mM for propranolol. The values of v max for chlorzoxazone and propranolol were 46.0 ± 9.0 and 7.6 ± 3.4 nmol min−1 nmol−1, respectively. These values are higher then those measured for the human enzymes. The v max value for aniline was 9.4 ± 1.3 nmol min−1 nmol−1, comparable to that calculated for human cytochromes P450. The functional data were found to be in line with the sequence alignments, showing that the identity percentage of CYP102A1 with CYP3A4 and CYP2E1 is higher than that found for CYP1A2, CYP2C9 and CYP2D6 families.

In this work the catalytic properties of a cytochrome P450 immobilised onto an electrode surface are improved by means of the molecular Lego approach.

The membrane-bound human cytochrome P450s have essential roles in the metabolism of endogenous compounds and drugs. Presented here are the results on the construction and characterization of three fusion proteins containing the N-terminally modified human cytochrome P450s CYP2C9, CY2C19 and CYP3A4 fused to the soluble NADPH-dependent oxidoreductase domain of CYP102A1 from Bacillus megaterium. The constructs, CYP2C9/BMR, CYP2C19/BMR and CYP3A4/BMR are well expressed in Escherichia coli as holo proteins. The chimeras can be purified in the absence of detergent and the purified enzymes are both active and correctly folded in the absence of detergent, as demonstrated by circular dichroism and functional studies. Additionally, in comparison with the parent P450 enzyme, these chimeras have greatly improved solubility properties. The chimeras are catalytically self-sufficient and present turnover rates similar to those reported for the native enzymes in reconstituted systems, unlike previously reported mammalian cytochrome P450 fusion proteins. Furthermore the specific activities of these chimeras are not dependent on the enzyme concentration present in the reaction buffer and they do not require the addition of accessory proteins, detergents or phospholipids to be fully active. The solubility, catalytic self-sufficiency and wild-type like activities of these chimeras would greatly simplify the studies of cytochrome P450 mediated drug metabolism in solution.

This communication reports the first electrochemical study of the human P450 2E1 either absorbed or covalently linked to different electrode surfaces. Glassy-carbon and gold electrodes gave reversible electrochemical signals of an active P450 2E1. Molecular modeling of the enzyme helped to rationalize the results. A monolayer coverage was obtained on gold modified with cystamine/maleimide that covalently linked surface accessible cysteines of P450 2E1. The midpoint potential measured for the oriented P450 2E1 was -177 +/- 5 mV comparable to that of the FeIII/FeII of other P450 enzymes. The observed electron-transfer rate for this electrode was 10 s-1. The turnover of the active enzyme was measured with the P450 2E1 specific substrate p-nitrophenol, resulting in a KM of 130 +/- 3 muM and the formation of 2.2 muM of the p-nitrocatechol product upon application of a -300 mV bias.

Cytochrome c-553 from Desulfovibrio vulgaris exhibits a highly exposed heme and an unusually low reduction potential with respect to other c-type cytochromes. Solvent heme exposure has been indicated as one of the most important factors in modulating the midpoint potential of the redox center. To test this hypothesis, a unique surface-exposed cysteine has been substituted for either M23 or G51 to produce the corresponding mutants and allow the formation of homodimers through a specific disulfide bridge. The reduction potentials, determined via spectroelectrochemistry, show an increase from +20 +/- 5 mV for the wt to +88 +/- 5 and +105 +/- 5 mV for the M23C-M23C homodimer and G51C-G51C homodimer, respectively. Chemical denaturation of the homodimers leads to parameters related to the hydrophobicity (m) and the number of buried side chains (n(B)), which suggest a decrease of exposure of the heme as a result of dimerization. These results are consistent with the heme-accessible surface area (ASA) calculated from a computer model of the homodimers. The ASA values show a decrease from 73 A(2) for the wt to 66 and 50 A(2) per heme for the M23C-M23C homodimer and G51C-G51C homodimer, respectively. The trend of the m- and n(B)-values, the degree of solvent accessibility, and the midpoint potential observed upon formation of the homodimers indicate a correlation between the reduction potential values and the exclusion of water from the heme surface.

Redox proteins and enzymes are attractive targets for nanobiotechnology. The theoretical framework of biological electron transfer is increasingly well-understood, and several properties make redox centres good systems for exploitation: many can be detected both electrochemically and optically; they can perform specific reactions; they are capable of self-assembly; and their dimensions are in the nanoscale. Great progress has been made with the two main approaches of protein engineering: rational design and combinatorial synthesis. Rational design has put our understanding of the structure–function relationship to the test, whereas combinatorial synthesis has generated new molecules of interest. This article provides selected examples of novel approaches where redox proteins are ‘wired up' in efficient electron-transfer chains, are ‘assembled' in artificial multidomain structures (molecular Lego), are ‘linked' to surfaces in nanodevices for biosensing and nanobiotechnological applications.

Measures to mitigate the emissions of carbon dioxide (CO2) can vary substantially in terms of the energy required. Some proposed CO2 mitigation options involve energy-intensive processes that compromise their viability as routes to mitigation, especially if deployed at a global scale. Here we provide an assessment of different mitigation options in terms of their energy usage. We assess the relative effectiveness of several CO2 mitigation routes by calculating the energy cost of carbon abatement (kilowatt-hour spent per kilogram CO2-equivalent, or kWh kgCO2e–1) mitigated. We consider energy efficiency measures, decarbonizing electricity, heat, chemicals and fuels, and also capturing CO2 from air. Among the routes considered, switching to renewable energy technologies (0.05–0.53 kWh kgCO2e–1 mitigated) offer more energy-effective mitigation than carbon embedding or carbon removal approaches, which are more energy intensive (0.99–10.03 kWh kgCO2e–1 and 0.78–2.93 kWh kgCO2e–1 mitigated, respectively), whereas energy efficiency measures, such as improving building lighting, can offer the most energy-effective mitigation. Carbon emission reduction measures have widely differing energy consumptions that have not been systematically compared. Babacan et al. estimate comparable energy use per unit emission reduction of various emission reduction measures, from efficiency improvements to renewable electricity generation to carbon removal.

Background: Retinol is tightly packed inside the structure of its plasma carrier (retinol-binding protein, RBP). It was found that retinol release from RBP to aqueous solutions is facilitated by either very low pH or very high temperatures (i.e. by non-physiological conditions that cause protein denaturation). It was also found that alcohols induce protein conformational transitions to denatured states. On this basis, it may be suggested that retinol release in vivo is facilitated by the partial unfolding of the carrier resulting from the concerted action of the moderate local decrease of pH and the moderate local decrease of dielectric constant in proximity to the target membranes. Results: In vitro , at 37°C, retinol is removed from its plasma carrier by the concerted action of the moderately low pH and the moderately low dielectric constant of solutions containing a low ionic strength buffer and methanol in variable proportions. Release of retinol is accompanied by a conformational transition of RBP from the native to the molten-globule state. Conclusions: The physiological function of RBP – targeted delivery of retinol – is mimicked in vitro by the facilitated release of retinol (associated with a partial unfolding of the protein carrier) in solutions exhibiting pH and dielectric constant values that are within the range of values expected in the in vivo microenvironment.

This work reports on a novel approach for building artificial redox chains: the molecular ‘Lego’ approach. This exploits the scaffold of natural redox proteins by fusing together functional protein modules with the desired properties. The molecular ‘Lego’ mimics the natural molecular evolution that proceeded by modular assembly of genes/DNA segments. Non-physiological electron transfer partners, flavodoxin (fld) and cytochrome c553 (c553) from Desulfoibrio ulgaris and the haem domain of P450 BM3 (BMP) from Bacillus megaterium have been used as building blocks in different combinations to build artificial redox chains. The kinetic characterisation of the electron transfer (ET) between the separate building blocks has been carried out. Under pseudo-first order conditions, a limiting ET rate, klim, of 0.48±0.05 s−1 and 43.77±2.18 s−1 and an apparent binding constant, Kapp, of 21±6 μM and 1.23±0.32 μM have been found for the fld/c553 and fld/BMP redox pairs, respectively. These results show that fld can be used as a module for transferring electrons to c553 and BMP. A 3D model of the fld/c553 and fld/BMP complexes was used to guide the construction of covalently linked assemblies ia engineered disulfide bridges or by fusion of the relevant genes ia an engineered loop. The first approach led to the construction, expression and characterisation of the S35C and S64C mutants of fld and M23C and G51C mutants of c553. Although the redox potentials of the separate mutants were found to be the same as those of recombinant wild type proteins (−408 mV for the semiquinone/hydroquinone couple of fld and +32 mV for the c553), the c553 homo-dimers M23C–M23C and G51C–G51C were found to have redox potentials of +88 and +105 mV, respectively. These differences have been analysed in terms of exposure of the haem cofactors to the solvent, and these lead to some interesting questions on the redox potentials of the transient redox complexes in physiological systems. The fld–c553 S64C–M23C and S35C–M23C chimeras were constructed, expressed and purified but the FMN was found to be destabilised resulting in the apo-form of these proteins. The gene fusion strategy was used to produce covalently linked assemblies of both fld–c553 and fld–BMP. The former was expressed using a seven amino acid (GPGPGPG) loop linking the C-terminus of fld to the N-terminus of c553. The fld–BMP fusion protein was successfully expressed by using the naturally occurring loop of the P450 BM3 (residues 471–479) to link the BMP domain at the N-terminus with fld domain at the C-terminus. This fusion was found to be correctly folded and functional. Efficient ET from the FMN to the haem domain (370 s−1) was also found to be in the same region of the physiological redox partners (250 s−1). This work demonstrates the feasibility of the molecular ‘ Lego’ approach in generating functional multi-domain proteins with designed properties, beyond the restrictions imposed by the naturally occurring protein domains.