Bicarbonate activation of the monomeric photosystem II-PsbS/Psb27 complex.

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.

Near ambient N2 fixation on solid electrodes versus enzymes and homogeneous catalysts.

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 revolutionize fertilizer production and even provide an energy-dense, truly carbon-free fuel. This Review provides a coherent comparison of recent progress made in dinitrogen fixation on solid electrodes, homogeneous catalysts and 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.

Impact of energy limitations on function and resilience in long-wavelength Photosystem II.

Photosystem II (PSII) uses the energy from red light to split water and reduce quinone, an energy-demanding process based on chlorophyll a (Chl-a) photochemistry. Two types of cyanobacterial PSII can use chlorophyll d (Chl-d) and chlorophyll f (Chl-f) to perform the same reactions using lower energy, far-red light. PSII from Acaryochloris marina has Chl-d replacing all but one of its 35 Chl-a, while PSII from Chroococcidiopsis thermalis, a facultative far-red species, has just 4 Chl-f and 1 Chl-d and 30 Chl-a. From bioenergetic considerations, the far-red PSII were predicted to lose photochemical efficiency and/or resilience to photodamage. Here, we compare enzyme turnover efficiency, forward electron transfer, back-reactions and photodamage in Chl-f-PSII, Chl-d-PSII, and Chl-a-PSII. We show that: (i) all types of PSII have a comparable efficiency in enzyme turnover; (ii) the modified energy gaps on the acceptor side of Chl-d-PSII favour recombination via PD1+Phe- repopulation, leading to increased singlet oxygen production and greater sensitivity to high-light damage compared to Chl-a-PSII and Chl-f-PSII; (iii) the acceptor-side energy gaps in Chl-f-PSII are tuned to avoid harmful back reactions, favouring resilience to photodamage over efficiency of light usage. The results are explained by the differences in the redox tuning of the electron transfer cofactors Phe and QA and in the number and layout of the chlorophylls that share the excitation energy with the primary electron donor. PSII has adapted to lower energy in two distinct ways, each appropriate for its specific environment but with different functional penalties.

Algae, plants and cyanobacteria perform a process called photosynthesis, in which carbon dioxide and water are converted into oxygen and energy-rich carbon compounds. The first step of this process involves an enzyme called photosystem II, which uses light energy to extract electrons from water to help capture the carbon dioxide. If the photosystem absorbs too much light, compounds known as reactive oxygen species are produced in quantities that damage the photosystem and kill the cell. To ensure that the photosystem works efficiently and to protect it from damage, about half of the energy from the absorbed light is dissipated as heat, while the rest of the energy is stored in the products of photosynthesis. The standard form of photosystem II uses the energy of visible light, but some cyanobacteria contain different types of photosystem II, which do the same chemical reactions using lower energy far-red light. One type of far-red photosystem II is found in Acaryochloris marina, a cyanobacterium living in stable levels of far-red light, shaded from visible light. The other type is found in a cyanobacterium called Chroococcidiopsis thermalis, which can switch between using its far-red photosystem II when shaded from visible light and using its standard photosystem II when exposed to it. Being able to work with less energy, the two types of far-red photosystem II appear to be more efficient than the standard one, but it has been unclear if there were any downsides to this trait. Viola et al. compared the standard photosystem II with the far-red photosystem II types from C. thermalis and A. marina by measuring the efficiency of these enzymes, the quantity of reactive oxygen species produced, and the resulting light-induced damage. The experiments revealed that the far-red photosystem II of A. marina is highly efficient but produces elevated levels of reactive oxygen species if exposed to high light conditions. On the other hand, the far-red photosystem II of C. thermalis is less efficient in collecting and using far-red light, but is more robust, producing fewer reactive oxygen species. Despite these tradeoffs, engineering crop plants or algae that could use far-red photosynthesis may help boost food and biomass production. A better understanding of the trade-offs between efficiency and resilience in the two types of far-red photosystem II could determine which features would be beneficial, and under what conditions. This work also improves our knowledge of how the standard photosystem II balances light absorption and damage limitation to work efficiently in a variable environment.

Bicarbonate-controlled reduction of oxygen by the QA semiquinone in Photosystem II in membranes.

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 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 [Formula: see text] 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 [Formula: see text], with an apparent dissociation constant of 70 ± 20 µM. Unexpectedly, [Formula: see text] 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 [Formula: see text] These results, together with molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics calculations, indicate that electron transfer from [Formula: see text] 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 [Formula: see text] 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 [Formula: see text] This could be beneficial by oxidizing [Formula: see text] and by producing superoxide, a chemical signal for the overreduced state of the electron transfer chain.

Femtosecond visible transient absorption spectroscopy of chlorophyll-f-containing photosystem II.

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.

The primary donor of far-red photosystem II: ChlD1 or 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 ±â€¯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.

Bicarbonate-Mediated CO2 Formation on Both Sides of Photosystem II.

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 QA quinone leads to the release of HCO3- from its binding site on the non-heme iron and (ii) the Em potential of the QA/QA•- couple increases when HCO3- dissociates. This suggested a protective/regulatory role of HCO3- as it is known that increasing the Em of QA decreases the extent of back-reaction-associated photodamage. Here we demonstrate, using plant thylakoids, that time-resolved membrane-inlet mass spectrometry together with 13C 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.

Energetics of the exchangeable quinone, QB, in Photosystem II.

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.

A low-potential terminal oxidase associated with the iron-only nitrogenase from the nitrogen-fixing bacterium Azotobacter vinelandii.

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.

Oxygenic Photoreactivity in Photosystem II Studied by Rotating Ring Disk Electrochemistry.

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.

Photochemistry beyond the red limit in chlorophyll f-containing photosystems.

Photosystems I and II convert solar energy into the chemical energy that powers life. Chlorophyll a photochemistry, using red light (680 to 700 nm), is near universal and is considered to define the energy "red limit" of oxygenic photosynthesis. We present biophysical studies on the photosystems from a cyanobacterium grown in far-red light (750 nm). The few long-wavelength chlorophylls present are well resolved from each other and from the majority pigment, chlorophyll a. Charge separation in photosystem I and II uses chlorophyll f at 745 nm and chlorophyll f (or d) at 727 nm, respectively. Each photosystem has a few even longer-wavelength chlorophylls f that collect light and pass excitation energy uphill to the photochemically active pigments. These photosystems function beyond the red limit using far-red pigments in only a few key positions.

Glycolate Induces Redox Tuning Of Photosystem II in Vivo: Study of a Photorespiration Mutant.

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 QA to a more positive potential and lowers the yield of singlet oxygen (1O2) production. The presence of QA- 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 1O2 in vivo under photorespiratory conditions. Here, we compared the properties of QA 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 QA and QB in the mutant. Glycolate induced an increase in the temperature maximum of thermoluminescence emission, indicating a shift of the midpoint potential of QA to a more positive value. The yield of 1O2 production was lowered in thylakoid membranes isolated from hpr1-1 compared with the wild type, consistent with a higher potential of QA/QA- 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-CO2 conditions.

Photoelectrochemistry of Photosystem II in Vitro vs in Vivo.

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 (<6 h), with turnover numbers surpassing those of the protein-film after 2 days. The mechanism of biofilm electrogenesis is suggested to involve an intracellular redox mediator, which is released during light irradiation.

Electricity generation from digitally printed cyanobacteria.

Microbial biophotovoltaic cells exploit the ability of cyanobacteria and microalgae to convert light energy into electrical current using water as the source of electrons. Such bioelectrochemical systems have a clear advantage over more conventional microbial fuel cells which require the input of organic carbon for microbial growth. However, innovative approaches are needed to address scale-up issues associated with the fabrication of the inorganic (electrodes) and biological (microbe) parts of the biophotovoltaic device. Here we demonstrate the feasibility of using a simple commercial inkjet printer to fabricate a thin-film paper-based biophotovoltaic cell consisting of a layer of cyanobacterial cells on top of a carbon nanotube conducting surface. We show that these printed cyanobacteria are capable of generating a sustained electrical current both in the dark (as a 'solar bio-battery') and in response to light (as a 'bio-solar-panel') with potential applications in low-power devices.

Bicarbonate-induced redox tuning in Photosystem II for regulation and protection.

The midpoint potential (Em) of [Formula: see text], the one-electron acceptor quinone of Photosystem II (PSII), provides the thermodynamic reference for calibrating PSII bioenergetics. Uncertainty exists in the literature, with two values differing by ∼80 mV. Here, we have resolved this discrepancy by using spectroelectrochemistry on plant PSII-enriched membranes. Removal of bicarbonate (HCO3-) shifts the Em from ∼-145 mV to -70 mV. The higher values reported earlier are attributed to the loss of HCO3- during the titrations (pH 6.5, stirred under argon gassing). These findings mean that HCO3- binds less strongly when QA-• is present. Light-induced QA-• formation triggered HCO3- loss as manifest by the slowed electron transfer and the upshift in the Em of QA HCO3--depleted PSII also showed diminished light-induced 1O2 formation. This finding is consistent with a model in which the increase in the Em of [Formula: see text] promotes safe, direct [Formula: see text] charge recombination at the expense of the damaging back-reaction route that involves chlorophyll triplet-mediated 1O2 formation [Johnson GN, et al. (1995) Biochim Biophys Acta 1229:202-207]. These findings provide a redox tuning mechanism, in which the interdependence of the redox state of QA and the binding by HCO3- regulates and protects PSII. The potential for a sink (CO2) to source (PSII) feedback mechanism is discussed.

Photocurrents from photosystem II in a metal oxide hybrid system: Electron transfer pathways.

We have investigated the nature of the photocurrent generated by Photosystem II (PSII), the water oxidizing enzyme, isolated from Thermosynechococcus elongatus, when immobilized on nanostructured titanium dioxide on an indium tin oxide electrode (TiO2/ITO). We investigated the properties of the photocurrent from PSII when immobilized as a monolayer versus multilayers, in the presence and absence of an inhibitor that binds to the site of the exchangeable quinone (QB) and in the presence and absence of exogenous mobile electron carriers (mediators). The findings indicate that electron transfer occurs from the first quinone (QA) directly to the electrode surface but that the electron transfer through the nanostructured metal oxide is the rate-limiting step. Redox mediators enhance the photocurrent by taking electrons from the nanostructured semiconductor surface to the ITO electrode surface not from PSII. This is demonstrated by photocurrent enhancement using a mediator incapable of accepting electrons from PSII. This model for electron transfer also explains anomalies reported in the literature using similar and related systems. The slow rate of the electron transfer step in the TiO2 is due to the energy level of electron injection into the semiconducting material being below the conduction band. This limits the usefulness of the present hybrid electrode. Strategies to overcome this kinetic limitation are discussed.

Iron-based redox centres of reductase and oxygenase components of phenol hydroxylase from A. radioresistens: a redox chain working at highly positive redox potentials.

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.

A new standardized electrochemical array for drug metabolic profiling with human cytochromes P450.

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.