A comprehensive assessment of photosynthetic acclimation to shade in C4 grass (Cynodon dactylon (L.) Pers.).

BACKGROUND: Light deficit in shaded environment critically impacts the growth and development of turf plants. Despite this fact, past research has predominantly concentrated on shade avoidance rather than shade tolerance. To address this, our study examined the photosynthetic adjustments of Bermudagrass when exposed to varying intensities of shade to gain an integrative understanding of the shade response of C4 turfgrass. RESULTS: We observed alterations in photosynthetic pigment-proteins, electron transport and its associated carbon and nitrogen assimilation, along with ROS-scavenging enzyme activity in shaded conditions. Mild shade enriched Chl b and LHC transcripts, while severe shade promoted Chl a, carotenoids and photosynthetic electron transfer beyond QA- (ET0/RC, φE0, Ψ0). The study also highlighted differential effects of shade on leaf and root components. For example, Soluble sugar content varied between leaves and roots as shade diminished SPS, SUT1 but upregulated BAM. Furthermore, we observed that shading decreased the transcriptional level of genes involving in nitrogen assimilation (e.g. NR) and SOD, POD, CAT enzyme activities in leaves, even though it increased in roots. CONCLUSIONS: As shade intensity increased, considerable changes were noted in light energy conversion and photosynthetic metabolism processes along the electron transport chain axis. Our study thus provides valuable theoretical groundwork for understanding how C4 grass acclimates to shade tolerance.

Respiration in light of evergreen and deciduous woody species and its links to the leaf economic spectrum.

Leaf respiration in the light (Rlight) is crucial for understanding the net CO2 exchange of individual plants and entire ecosystems. However, Rlight is poorly quantified and rarely discussed in the context of the leaf economic spectrum (LES), especially among woody species differing in plant functional types (PFTs) (e.g., evergreen vs. deciduous species). To address this gap in our knowledge, Rlight, respiration in the dark (Rdark), light-saturated photosynthetic rates (Asat), leaf dry mass per unit area (LMA), leaf nitrogen (N) and phosphorus (P) concentrations, and maximum carboxylation (Vcmax) and electron transport rates (Jmax) of 54 representative subtropical woody evergreen and deciduous species were measured. With the exception of LMA, the parameters quantified in this study were significantly higher in deciduous species than in evergreen species. The degree of light inhibition did not significantly differ between evergreen (52%) and deciduous (50%) species. Rlight was significantly correlated with LES traits such as Asat, Rdark, LMA, N and P. The Rlight vs. Rdark and N relationships shared common slopes between evergreen and deciduous species, but significantly differed in their y-intercepts, in which the rates of Rlight were slower or faster for any given Rdark or N in deciduous species, respectively. A model for Rlight based on three traits (i.e., Rdark, LMA and P) had an explanatory power of 84.9%. These results show that there is a link between Rlight and the LES, and highlight that PFTs is an important factor in affecting Rlight and the relationships of Rlight with Rdark and N. Thus, this study provides information that can improve the next generation of terrestrial biosphere models (TBMs).

Protein charge transfer far from equilibrium: a theoretical perspective.

Potential differences for protein-assisted electron transfer across lipid bilayers or in bio-nano setups can amount to several 100 mV; they lie far outside the range of linear response theory. We describe these situations by Pauli-master equations that are based on Marcus theory of charge transfer between self-trapped electrons and that obey Kirchhoff's current law. In addition, we take on-site blockade effects and a full non-linear response of the local potentials into account. We present analytical and numerical current-potential curves and electron populations for multi-site model systems and biological electron transfer chains. Based on these, we provide empirical rules for electron populations and chemical potentials along the chain. The Pauli-master mean-field results are validated by kinetic Monte Carlo simulations. We briefly discuss the biochemical and evolutionary aspects of our findings.

Application of biochar in microbial fuel cells: Characteristic performances, electron-transfer mechanism, and environmental and economic assessments.

Biochar is a by-product of thermochemical conversion of biomass or other carbonaceous materials. Recently, it has garnered extensive attention for its high application potential in microbial fuel cell (MFC) systems owing to its high conductivity and low cost. However, the effects of biochar on MFC system performance have not been comprehensively reviewed, thereby necessitating the evaluation of the efficacy of biochar application in MFCs. In this review, biochar characteristics were outlined based on recent publications. Subsequently, various applications of biochar in the MFC systems and their probable processes were summarized. Finally, proposals for future applications of biochar in MFCs were explored along with its perspectives and an environmental evaluation in the context of a circular economy. The purpose of this review is to gain comprehensive insights into the application of biochar in the MFC systems, offering important viewpoints on the effective and steady utilization of biochar in MFCs for practical application.

Assessment of the recovery and photosynthetic efficiency of Breviolum psygmophilum and Effrenium voratum (Symbiodiniaceae) following cryopreservation.

Many strains of Symbiodiniaceae have been isolated and their genetics, taxonomy, and metabolite production studied. Maintaining these cultures requires careful and regular sub-culturing that is costly with a high risk of species contamination or loss. Cryopreservation is a viable alternative for their long-term storage; however, there is uncertainty as to whether cryopreservation impacts the photosynthetic performance of Symbiodiniaceae. We investigated the growth rates and photosynthetic efficiency of two species, Breviolum psygmophilum and Effrenium voratum before and after cryopreservation. Rapid light curves (RLCs) produced using Pulse Amplitude Modulated (PAM) fluorometry were used to generate detailed information on the characteristics of photosystem II (PSII). The maximum electron transport rate (ETRmax) and the quantum yield (Fv/Fm) of the control (non-cryopreserved) and cryopreserved culture isolates were assessed across the growth cycle. The non-cryopreserved isolate of B. psygmophilum had a higher quantum yield than the cryopreserved isolate from day 12 to day 24, whereas there were no differences from day 28 to the late stationary phase. There were no significant differences in ETRmax. No significant differences were observed in quantum yield or ETRmax between the control and cryopreserved E. voratum isolates. The ability of cryopreserved strains to recover and regain their photosynthetic efficiency after freezing demonstrates the utility of this method for the long-term storage of these and other Symbiodiniaceae species.

Carbon Nanotube-Based Biosensors Using Fusion Technologies with Biologicals & Chemicals for Food Assessment.

High-sensitivity sensors applied in various diagnostic systems are considered to be a promising technology in the era of the fourth industrial revolution. Biosensors that can quickly detect the presence and concentration of specific biomaterials are receiving research attention owing to the breakthroughs in detection technology. In particular, the latest technologies involving the miniaturization of biosensors using nanomaterials, such as nanowires, carbon nanotubes, and nanometals, have been widely studied. Nano-sized biosensors applied in food assessment and in in vivo measurements have the advantages of rapid diagnosis, high sensitivity and selectivity. Nanomaterial-based biosensors are inexpensive and can be applied to various fields. In the present society, where people are paying attention to health and wellness, high-technology food assessment is becoming essential as the consumer demand for healthy food increases. Thus, biosensor technology is required in the food and medical fields. Carbon nanotubes (CNTs) are widely studied for use in electrochemical biosensors. The sensitive electrical characteristics of CNTs allow them to act as electron transfer mediators in electrochemical biosensors. CNT-based biosensors require novel technologies for immobilizing CNTs on electrodes, such as silicon wafers, to use as biosensor templates. CNT-based electrochemical biosensors that serve as field-effect transistors (FET) increase sensitivity. In this review, we critically discuss the recent advances in CNT-based electrochemical biosensors applied with various receptors (antibodies, DNA fragments, and other nanomaterials) for food evaluation, including pathogens, food allergens, and other food-based substances.

Investigation of Monte Carlo simulations of the electron transport in external magnetic fields using Fano cavity test.

PURPOSE: Monte Carlo simulations are crucial for calculating magnetic field correction factors kB for the dosimetry in external magnetic fields. As in Monte Carlo codes the charged particle transport is performed in straight condensed history (CH) steps, the curved trajectories of these particles in the presence of external magnetic fields can only be approximated. In this study, the charged particle transport in presence of a strong magnetic field B→ was investigated using the Fano cavity test. The test was performed in an ionization chamber and a diode detector, showing how the step size restrictions must be adjusted to perform a consistent charged particle transport within all geometrical regions. METHODS: Monte Carlo simulations of the charged particle transport in a magnetic field of 1.5 T were performed using the EGSnrc code system including an additional EMF-macro for the transport of charged particle in electro-magnetic fields. Detailed models of an ionization chamber and a diode detector were placed in a water phantom and irradiated with a so called Fano source, which is a monoenergetic, isotropic electron source, where the number of emitted particles is proportional to the local density. RESULTS: The results of the Fano cavity test strongly depend on the energy of charged particles and the density within the given geometry. By adjusting the maximal length of the charged particle steps, it was possible to calculate the deposited dose in the investigated regions with high accuracy (<0.1%). The Fano cavity test was performed in all regions of the detailed detector models. Using the default value for the step size in the external magnetic field, the maximal deviation between Monte Carlo based and analytical dose value in the sensitive volume of the ion chamber and diode detector was 8% and 0.1%, respectively. CONCLUSIONS: The Fano cavity test is a crucial validation method for the modeled detectors and the transport algorithms when performing Monte Carlo simulations in a strong external magnetic field. Special care should be given, when calculating dose in volumes of low density. This study has shown that the Fano cavity test is a useful method to adapt particle transport parameters for a given simulation geometry.

The Biochemical Assessment of Mitochondrial Respiratory Chain Disorders.

Mitochondrial respiratory chain (MRC) disorders are a complex group of diseases whose diagnosis requires a multidisciplinary approach in which the biochemical investigations play an important role. Initial investigations include metabolite analysis in both blood and urine and the measurement of lactate, pyruvate and amino acid levels, as well as urine organic acids. Recently, hormone-like cytokines, such as fibroblast growth factor-21 (FGF-21), have also been used as a means of assessing evidence of MRC dysfunction, although work is still required to confirm their diagnostic utility and reliability. The assessment of evidence of oxidative stress may also be an important parameter to consider in the diagnosis of MRC function in view of its association with mitochondrial dysfunction. At present, due to the lack of reliable biomarkers available for assessing evidence of MRC dysfunction, the spectrophotometric determination of MRC enzyme activities in skeletal muscle or tissue from the disease-presenting organ is considered the 'Gold Standard' biochemical method to provide evidence of MRC dysfunction. The purpose of this review is to outline a number of biochemical methods that may provide diagnostic evidence of MRC dysfunction in patients.

Assessment of the Photosynthetic Apparatus Functions by Chlorophyll Fluorescence and P700 Absorbance in C3 and C4 Plants under Physiological Conditions and under Salt Stress.

Functions of the photosynthetic apparatus of C3 (Pisum sativum L.) and C4 (Zea mays L.) plants under physiological conditions and after treatment with different NaCl concentrations (0-200 mM) were investigated using chlorophyll a fluorescence (pulse-amplitude-modulated (PAM) and JIP test) and P700 photooxidation measurement. Data revealed lower density of the photosynthetic structures (RC/CSo), larger relative size of the plastoquinone (PQ) pool (N) and higher electron transport capacity and photosynthetic rate (parameter RFd) in C4 than in C3 plants. Furthermore, the differences were observed between the two studied species in the parameters characterizing the possibility of reduction in the photosystem (PSI) end acceptors (REo/RC, REo/CSo and δRo). Data revealed that NaCl treatment caused a decrease in the density of the photosynthetic structures and relative size of the PQ pool as well as decrease in the electron transport to the PSI end electron acceptors and the probability of their reduction as well as an increase in the thermal dissipation. The effects were stronger in pea than in maize. The enhanced energy losses after high salt treatment in maize were mainly from the increase in the regulated energy losses (ΦNPQ), while in pea from the increase in non-regulated energy losses (ΦNO). The reduction in the electron transport from QA to the PSI end electron acceptors influenced PSI activity. Analysis of the P700 photooxidation and its decay kinetics revealed an influence of two PSI populations in pea after treatment with 150 mM and 200 mM NaCl, while in maize the negligible changes were registered only at 200 mM NaCl. The experimental results clearly show less salt tolerance of pea than maize.

Characterizing Protein Protonation Microstates Using Monte Carlo Sampling.

Proteins are polyelectrolytes with acidic and basic amino acids Asp, Glu, Arg, Lys, and His, making up ≈25% of the residues. The protonation state of residues, cofactors, and ligands defines a "protonation microstate". In an ensemble of proteins some residues will be ionized and others neutral, leading to a mixture of protonation microstates rather than in a single one as is often assumed. The microstate distribution changes with pH. The protein environment also modifies residue proton affinity so microstate distributions change in different reaction intermediates or as ligands are bound. Particular protonation microstates may be required for function, while others exist simply because there are many states with similar energy. Here, the protonation microstates generated in Monte Carlo sampling in MCCE are characterized in HEW lysozyme as a function of pH and bacterial photosynthetic reaction centers (RCs) in different reaction intermediates. The lowest energy and highest probability microstates are compared. The ΔG, ΔH, and ΔS between the four protonation states of Glu35 and Asp52 in lysozyme are shown to be calculated with reasonable precision. At pH 7 the lysozyme charge ranges from 6 to 10, with 24 accepted protonation microstates, while RCs have ≈50,000. A weighted Pearson correlation analysis shows coupling between residue protonation states in RCs and how they change when the quinone in the QB site is reduced. Protonation microstates can be used to define input MD parameters and provide insight into the motion of protons coupled to reactions.

Simulating the Feasibility of Using Liquid Micro-Jets for Determining Electron-Liquid Scattering Cross-Sections.

The extraction of electron-liquid phase cross-sections (surface and bulk) is proposed through the measurement of (differential) energy loss spectra for electrons scattered from a liquid micro-jet. The signature physical elements of the scattering processes on the energy loss spectra are highlighted using a Monte Carlo simulation technique, originally developed for simulating electron transport in liquids. Machine learning techniques are applied to the simulated electron energy loss spectra, to invert the data and extract the cross-sections. The extraction of the elastic cross-section for neon was determined within 9% accuracy over the energy range 1-100 eV. The extension toward the simultaneous determination of elastic and ionisation cross-sections resulted in a decrease in accuracy, now to within 18% accuracy for elastic scattering and 1% for ionisation. Additional methods are explored to enhance the accuracy of the simultaneous extraction of liquid phase cross-sections.

Prediction of Monomeric and Dimeric Structures of CYP102A1 Using AlphaFold2 and AlphaFold Multimer and Assessment of Point Mutation Effect on the Efficiency of Intra- and Interprotein Electron Transfer.

The three-dimensional structure of monomers and homodimers of CYP102A1/WT (wild-type) proteins and their A83F and A83I mutant forms was predicted using the AlphaFold2 (AF2) and AlphaFold Multimer (AFMultimer) programs, which were compared with the rate constants of hydroxylation reactions of these enzyme forms to determine the efficiency of intra- and interprotein electron transport in the CYP102A1 hydroxylase system. The electron transfer rate constants (ket), which determine the rate of indole hydroxylation by the CYP102A1 system, were calculated based on the distances (R) between donor-acceptor prosthetic groups (PG) FAD→FMN→HEME of these proteins using factor ß, which describes an exponential decay from R the speed of electron transport (ET) according to the tunnelling mechanism. It was shown that the structure of monomers in the homodimer, calculated using the AlpfaFold Multimer program, is in good agreement with the experimental structures of globular domains (HEME-, FMN-, and FAD-domains) in CYP102A1/WT obtained by X-ray structural analysis, and the structure of isolated monomers predicted in AF2 does not coincide with the structure of monomers in the homodimer, although a high level of similarity in individual domains remains. The structures of monomers and homodimers of A83F and A83I mutants were also calculated, and their structures were compared with the wild-type protein. Significant differences in the structure of all isolated monomers with respect to the structures of monomers in homodimers were also found for them, and at the same time, insignificant differences were revealed for all homodimers. Comparative analysis for CYP102A1/WT between the calculated intra- and interprotein distances FAD→FMN→HEME and the rate constants of hydroxylation in these proteins showed that the distance between prosthetic groups both in the monomer and in the dimer allows the implementation of electron transfer between PGs, which is consistent with experimental literature data about kcat. For the mutant form of monomer A83I, an increase in the distance between PGs was obtained, which can restrict electron transportation compared to WT; however, for the dimer of this protein, a decrease in the distance between PGs was observed compared to the WT form, which can lead to an increase in the electron transfer rate constant and, accordingly, kcat. For the monomer and homodimer of the A83F mutant, the calculations showed an increase in the distance between the PGs compared to the WT form, which should have led to a decrease in the electron transfer rate, but at the same time, for the homodimer, the approach of the aromatic group F262 with heme can speed up transportation for this form and, accordingly, the rate of hydroxylation.

Enhancing the population of the encounter complex affects protein complex formation efficiency.

Optimal charge distribution is considered to be important for efficient formation of protein complexes. Electrostatic interactions guide encounter complex formation that precedes the formation of an active protein complex. However, disturbing the optimized distribution by introduction of extra charged patches on cytochrome c peroxidase does not lead to a reduction in productive encounters with its partner cytochrome c. To test whether a complex with a high population of encounter complex is more easily affected by suboptimal charge distribution, the interactions of cytochrome c mutant R13A with wild-type cytochrome c peroxidase and a variant with an additional negative patch were studied. The complex of the peroxidase and cytochrome c R13A was reported to have an encounter state population of 80%, compared to 30% for the wild-type cytochrome c. NMR analysis confirms the dynamic nature of the interaction and demonstrates that the mutant cytochrome c samples the introduced negative patch. Kinetic experiments show that productive complex formation is fivefold to sevenfold slower at moderate and high ionic strength values for cytochrome c R13A but the association rate is not affected by the additional negative patch on cytochrome c peroxidase, showing that the total charge on the protein surface can compensate for less optimal charge distribution. At low ionic strength (44 mm), the association with the mutant cytochrome c reaches the same high rates as found for wild-type cytochrome c, approaching the diffusion limit.

Evaluation of the electron transport algorithm in magnetic field in EGS5 Monte Carlo code.

PURPOSE: To evaluate the accuracy of electron transport in the magnetic field of Electron Gamma Shower version 5 (EGS5) by using the special Fano cavity test. METHODS: To simulate electron transport in the magnetic field, the trajectory of the electron was reconstructed with a short step length to restrict fractional energy loss, and the maximum user step length (mxustep) was set at 0.01 cm or 0.001 cm. For the special Fano cavity test, three-layer slab Fano test geometry was used, and uniform and isotropic per unit mass mono-energetic electrons with 0.01, 0.1, 1.0, and 10 MeV were permitted from the central axis of geometry in 0.35 T and 1.5 T. Furthermore, the magnetic field strength was scaled based on the mass density of the material. The relative difference between the calculated dose to gap and the theoretical value was evaluated. Furthermore, the special Fano cavity test was also performed using EGSnrc with the electron-enhanced electric and magnetic field macros under the same conditions, and the results were compared with those of EGS5. RESULTS: Deviations in 0.35 T were within 0.3% regardless of the parameter settings. In 1.5 T, stable results within 0.3% were obtained using 0.001 cm as the mxustep, except for one at 10 MeV. Further, the accuracy of EGSnrc was within 0.2%, except for 10 MeV for a 0.2-cm gap in 1.5 T. CONCLUSIONS: EGS5 with the appropriate parameter settings enable electron transport in magnetic fields similar with the accuracy of EGSnrc.

Assessment of Mitochondrial Cell Metabolism by Respiratory Chain Electron Flow Assays.

Cellular energy metabolism is regulated by complex metabolic pathways. Although anaerobic glycolysis was reported as a primary source of energy in cancer leading to a high rate of lactate production, current evidence shows that the main energy source supporting cancer cell metabolism relies on mitochondrial metabolism. Mitochondria are the key organelle maintaining optimal cellular energy levels. MitoPlate™ S-1 provides a highly reproducible bioenergetics tool to analyze the electron flow rate in live cells. Measuring the rates of electron flow into and through the electron transport chain using different NADH and FADH2-producing metabolic substrates enables the assessment of mitochondrial functionality. MitoPlate™ S-1 are 96-well microplates pre-coated with different substrates used as probes to examine the activity of mitochondrial metabolic pathways based on a colorimetric assay. A comparative metabolic analysis between cell lines or primary cells allows to establish a specific metabolic profile and to detect possible alterations of the mitochondrial function of a tumor cell. Moreover, the direct measurements of electron flux triggered by metabolic pathway activation could highlight targets for potential drug candidates.

In Silico Modeling of the Mitochondrial Pumping Complexes with Markov State Models.

The mechanism of proton pumping by the mitochondrial electron transport chain complexes is still enigmatic after decades of research. Recently, there has been interest in in silico Markov state models to model the mitochondrial pumping complexes at the microscopic level, and this chapter describes the methods of constructing and simulating such models.

The Charge Distribution on a Protein Surface Determines Whether Productive or Futile Encounter Complexes Are Formed.

Protein complex formation depends strongly on electrostatic interactions. The distribution of charges on the surface of redox proteins is often optimized by evolution to guide recognition and binding. To test the degree to which the electrostatic interactions between cytochrome c peroxidase (CcP) and cytochrome c (Cc) are optimized, we produced five CcP variants, each with a different charge distribution on the surface. Monte Carlo simulations show that the addition of negative charges attracts Cc to the new patches, and the neutralization of the charges in the regular, stereospecific binding site for Cc abolishes the electrostatic interactions in that region entirely. For CcP variants with the charges in the regular binding site intact, additional negative patches slightly enhance productive complex formation, despite disrupting the optimized charge distribution. Removal of the charges in the regular binding site results in a dramatic decrease in the complex formation rate, even in the presence of highly negative patches elsewhere on the surface. We conclude that additional charge patches can result in either productive or futile encounter complexes, depending on whether negative residues are located also in the regular binding site.

Assessment of the Ab Initio Bethe-Salpeter Equation Approach for the Low-Lying Excitation Energies of Bacteriochlorophylls and Chlorophylls.

Bacteriochlorophyll and chlorophyll molecules are crucial building blocks of the photosynthetic apparatus in bacteria, algae, and plants. Embedded in transmembrane protein complexes, they are responsible for the primary processes of photosynthesis: excitation energy and charge transfer. Here, we use ab initio many-body perturbation theory within the GW approximation and Bethe-Salpeter equation (BSE) approach to calculate the electronic structure and optical excitations of bacteriochlorophylls a, b, c, d, and e and chlorophylls a and b. We systematically study the effects of the structure, basis set size, partial self-consistency in GW, and the underlying exchange-correlation approximation and compare our calculations with results from time-dependent density functional theory, multireference RASPT2, and experimental literature results. We find that optical excitations calculated with GW+BSE are in excellent agreement with experimental data, with an average deviation of less than 100 meV for the first three bright excitations of the entire family of (bacterio)chlorophylls. Contrary to state-of-the-art time-dependent density functional theory (TDDFT) with an optimally tuned range-separated hybrid functional, this accuracy is achieved in a parameter-free approach. Moreover, GW+BSE predicts the energy differences between the low-energy excitations correctly and eliminates spurious charge transfer states that TDDFT with (semi)local approximations is known to produce. Our study provides accurate reference results and highlights the potential of the GW+BSE approach for the simulation of larger pigment complexes.

Assessment of the in vivo genotoxicity of pendimethalin via mitochondrial bioenergetics and transcriptional profiles during embryogenesis in zebrafish: Implication of electron transport chain activity and developmental defects.

Pendimethalin, an herbicide used to control weeds, acts by inhibiting plant cell division and mitosis. Several studies have reported the detrimental effects of pendimethalin on non-target organisms. It has been found to be especially toxic to aquatic life. Additionally, there is some evidence that pendimethalin induces mitochondrial stress. However, none of the studies have provided information about the functional defects in mitochondria and toxicity during embryogenesis. In this study, we evaluated the impact of pendimethalin on the electron transport chain (ETC) activity and mitochondrial complexes via in vivo screening of oxidative phosphorylation and transcriptional profiles in zebrafish embryos. The results showed that pendimethalin interferes with mitochondrial complexes I and V, which inhibit embryo energy metabolism, thereby leading to developmental defects. Transgenic zebrafish, fli1:eGFP and olig2:dsRed, were used to confirm pendimethalin-induced functional depletion in neurogenesis and vasculogenesis during embryo development. This study provides new insights into the methodology of environmental assessment of biohazard chemicals that target ETC activity in mitochondria. Additionally, the results suggest that real-time respiratory and metabolic monitoring in zebrafish will be useful for the genotoxicity assessment of environmentally hazardous substances and may be used as an alternative model for the control of aquatic environmental pollutants.

Assessment of the Maximal Activity of Complex IV in the Inner Mitochondrial Membrane by Tandem Electrochemistry and Respirometry.

Assessment of activities of mitochondrial electron transport enzymes is important for understanding mechanisms of metabolic diseases, but structural organization of mitochondria and low sample availability pose distinctive challenges for in situ functional studies. We report the development of a tandem microfluidic respirometer that simultaneously tracks both the reduction of mediators on the electrode and the ensuing reduction of O2 by complex IV in the inner mitochondrial membrane. The response time of O2 consumption to multiple alternating potential steps is of approximately 10 s for a 150 µm-thick sample. Steady O2 depletion shows good quantitative correlation with the supplied electric charge, Pearson's r = 0.994. Reduction of mediators on biocompatible gold electrodes modified with carbon ink or fumed silica can compete with the oxidation of mediators by mitochondria, yielding an overall respiratory activity comparable to that upon chemical reduction by ascorbate. The dependence of O2 consumption on mediator and mitochondrial suspension concentrations shows that mass transport between the electrode and mitochondria does not limit biological activity of the latter. The mediated electrochemical approach is validated by the radiometric measurements of simulated changes in the intrinsic mitochondrial activity upon partial inhibition of complex IV by NaN3. This approach enables the development of O2-independent, biomimetic electrochemical assays narrowly targeting components of the electron transport chains in their native environments.