Biochimica et Biophysica Acta (BBA) - Bioenergetics

Succinate dehydrogenase is an inner mitochondrial membrane protein complex that links the tricarboxylic acid cycle to the electron transport system. It catalyzes the reaction between succinate and ubiquinone to produce fumarate and ubiquinol. In addition, it can produce significant amounts of superoxide and hydrogen peroxide under the right conditions. While the flavin adenine dinucleotide (FAD) is the putative site of reactive oxygen species production, free radical production from other sites are less certain. Herein, we developed a computational model to analyze free radical production data from complex II and identify the mechanism of superoxide and hydrogen peroxide production. The model includes the major redox centers consisting of the FAD, three iron-sulfur clusters, and a transiently catalytic bound semi quinone. The model consists of five-states that represent oxidation status of the enzyme complex. Each step in the reaction scheme is thermodynamically constrained, and transitions between each state involve either one-electron or two-electron redox reactions. The model parameters were simultaneously fit using data consisting of enzyme kinetics and free radical production rates under a range of conditions. In the absence of respiratory chain inhibitors, model analysis revealed that the 3Fe-4S iron-sulfur cluster is the primary source of superoxide production followed by the FAD radical. However, when the quinone reductase site of complex II is inhibited or the quinone pool is highly reduced, superoxide production from the FAD site dominates at low succinate concentrations. In addition, hydrogen peroxide formation from the complex is only significant when these one of these conditions is met and the fumarate concentrations is in the low micromolar range. From the model simulations, the redox state of the quinone pool was found to be the primary determinant of free radical production from complex II. This study highlights the importance of evaluating enzyme kinetics and associated side-reactions in a consistent, quantitative and biophysical detailed manner. By incorporating the results from a diverse set of experiments, this computational approach can be used to interpret and explain key differences among the observations from a single, unified perspective.

The assembly of the Mn4O5Ca cluster of the photosystem II (PSII) starts from the initial binding and photooxidation of the first Mn2+ at a high affinity site (HAS). Recent cryo-EM apo-PSII structures reveal an altered geometry of amino ligands in this region and suggest the involvement of D1-Glu189 ligand in the formation of the HAS. We now find that Gln and Lys substitution mutants photoactivate with reduced quantum efficiency compared to the wild-type. However, the affinity of Mn2+ at the HAS in D1-E189K was very similar to the wild-type (~2.2 M). Thus, we conclude that D1-E189 does not form the HAS (~2.9 M) and that the reduced quantum efficiency of photoactivation in D1-E189K cannot be ascribed to the initial photooxidation of Mn2+ at the HAS. Besides reduced quantum efficiency, the D1-E189K mutant exhibits a large fraction of centers that fail to recover activity during photoactivation starting early in the assembly phase, becoming recalcitrant to further assembly. Fluorescence relaxation kinetics indicate on the presence of an alternative route for the charge recombination in Mn-depleted samples in all studied mutants and exclude damage to the photochemical reaction center as the cause for the recalcitrant centers failing to assemble and show that dark incubation of cells reverses some of the inactivation. This reversibility would explain the ability of these mutants to accumulate a significant fraction of active PSII during extended periods of cell growth. The failed recovery in the fraction of inactive centers appears to a reversible mis-assembly involving the accumulation of photooxidized, but non-catalytic high valence Mn at the donor side of photosystem II, and that a reductive mechanism exists for restoration of assembly capacity at sites incurring mis-assembly. Given the established role of Ca2+ in preventing misassembled Mn, we conclude that D1-E189K mutant impairs the ligation of Ca2+ at its effector site in all PSII centers that consequently leads to the mis-assembly resulting in accumulation of non-catalytic Mn at the donor side of PSII. Our data indicate that D1-E189 is not functionally involved in Mn2+ oxidation\binding at the HAS but rather involved in Ca2+ ligation and steps following the initial Mn2+ photooxidation.

Experimental assessment and computational modeling were used to analyze substrate transport, tricarboxylic acid cycle kinetics, and oxidative phosphorylation in suspensions of purified cardiac mitochondria. The kinetics of ATP synthesis and carbohydrate oxidation, including during hypoxia and reoxygenation, were investigated using various substrate combinations and conditions. Model simulations fit to transient respiration and NAD(P)H measurements reveal novel insights into pyruvate dehydrogenase regulation, regulation of mitochondrial leak, and the clearance of oxaloacetate during respiration on succinate. High concentrations of succinate induced increased mitochondrial leak respiration driven in part by ROS-activated uncoupling. Oxidative phosphorylation under succinate-fueled respiration was inhibited by rapid buildup of oxaloacetate, inhibiting succinate dehydrogenase. Malic enzyme and oxaloacetate decarboxylase activities represent potenital pathways for removal of oxaloacetate, with glutamate further enhancing clearance. The developed model captures the observed transient behaviors as well as steady-state relationships between ATP synthesis rate and phosphate metabolite levels, lending a new systems-level understanding of mitochondrial energy metabolism. In sum, these findings offer a framework for simulating and interpreting mitochondrial function in vitro and in vivo. Key PointsThis study uses experiments and computer simulations to probe the interactions between substrate transport processes, TCA cycle kinetics, redox state, and oxidative ATP synthesis in cardiac mitochondria. The developed kinetic model simulates mitochondrial metabolism in vitro and represents a framework for integrative modeling of cardiac energy metabolism. Model-based analysis identifies a kinetic model of pyruvate dehydrogenase (PDH) deactivation during leak-state respiration and activation during oxidative phosphorylation. High levels of cation leak during respiration on succinate are explained by a ROS-dependent activation of uncoupling.

The effects of TyrD*, TyrZ*, and QA*- radical formation on the absorption spectrum in the Soret region were studied in Mn-depleted Photosystem II at pH 8.6 (in order to be in the TyrD state after dark adaptation). Flash-induced difference spectra were recorded in several PSII samples from: i) Thermosynechococcus vestitus (formerly T. elongatus), ii) Synechocystis sp. PCC 6803, iii) Chroococcidiopsis thermalis PCC 7203 grown under far-red light, and iv) Acaryochloris marina. In the case of T. vestitus, mutants D1/H198Q, D1/T1789H, D2/I178H, and D2/Y160F, with PsbA1/Q130 instead of PsbA3/E130, were also studied for possible contributions from PD1, ChlD1, ChlD2, and PheD1, respectively. For a possible contribution from PD2, the D2/H197A mutant was studied in S. 6803. While PD1 is clearly the species whose spectrum is blue-shifted by [~]3nm in the presence of QA*-, as has already been well documented in the literature, the species whose spectra shift upon the formation of TyrD* and TyrZ* remain to be clearly identified, as they appear different from PD1, PD2, PheD1, ChlD1, and ChlD2, as concluded by the lack of different light-induced difference spectra in the mutants listed above. Although we cannot rule out a weak effect, considering the accuracy of the experiments, it is proposed that other pigments, such as antenna Chl and/or Car, near the reaction center are involved. Additionally, it is shown that: i) there is no proton release into the bulk upon the oxidation of TyrD at pH 8.6, and ii) the rearrangement of the electrostatic environment of the pigments involved in the light-induced difference spectra in the samples studied, upon the formation of TyrD*, TyrZ*, and QA*-, likely occurs differently from both a kinetic and structural perspective.

AbstractHuman NQO1 is a flavoenzyme essential for the redox metabolization of many substances and associated with wide-impacting diseases such as cancer and Alzheime[r]s. Recent X-ray crystallographic studies have proposed that a few residues at the active site of NQO1 (including Tyr126 and Tyr128) may control enzyme catalysis and functional negative cooperativity. In this work, we use rapid mixing pre-steady state kinetics and hydrogen-deuterium exchange followed by mass spectrometry (HDX-MS) to evaluate experimentally the role of Tyr126 and Tyr128 in NQO1 functionality by generating mutants to Phe, Ala and Glu. Mutations to Phe caused mild effects, whereas those to Ala significantly decreased hydride transfer efficiency and those to Glu virtually abolished NQO1 activity. Interestingly, structural stability studies by HDX-MS showed significant perturbations particularly affecting the binding site of NADH/NAD+ in the less conservative mutations (particularly to Glu). Mutations of Tyr126 and Tyr128 seem to also modulate the non-synchronous catalysis in the two active sites (negative cooperativity) as well as the selectivity for NADH/NADPH as coenzymes. Our work experimentally demonstrates the critical role of Tyr126 and Tyr128 in the flavin reductive half-reaction of the catalytic cycle of NQO1 in the negative cooperativity, and also suggests that phosphorylation of these two Tyr residues might shut down NQO1 activity reversibly.

The uptake of inorganic carbon in cyanobacteria is facilitated by an energetically intensive CO2-concentrating mechanism (CCM). Specialized Type-1 NDH complexes function as a part of this mechanism to couple photosynthetic energy generated by redox reactions of the electron transport chain (ETC) to CO2 hydration. This active site of CO2 hydration incorporates an arginine side chain as a Zn ligand, diverging from the typical histidine and/or cysteine residues found in standard CAs. In this study, we focused on mutating three amino acids in the active site of the constitutively expressed NDH-14 CO2 hydration complex in Synechococcus sp. PCC7942: CupB-R91, which acts as a zinc ligand, and CupB-E95 and CupB-H89, both of which are in close interaction with the arginine ligand. These mutations aimed to explore how they affect the unusual metal ligation by CupB-R91 and potentially influence the unusual catalytic process. The most severe defects in activity among the targeted residues are due to a substitution of CupB-R91 and the ionically interacting E95 since both proved essential for the structural stability of the CupB protein. On the other hand, CupB-H89 mutations show a range of catalytic phenotypes indicating a role of this residue in the catalytic mechanism of CO2-hydration, but no evidence was obtained for aberrant carbonic anhydrase activity that would have indicated uncoupling of the CO2-hydration activity from proton pumping. The results are discussed in terms of possible alternative CO2 hydration mechanisms.

Binding of Psb28 to the photosystem II assembly intermediate PSII-I induces conformational changes to the PSII acceptor side that impact charge recombination and reduce the in situ production of singlet oxygen (Zabret et al. 2021, Nat. Plants 7, 524-538). A detailed fluorometric analysis of the PSII-I assembly intermediate compared with OEC-disrupted and Mn-depleted PSII complexes showed differences between their variable (OJIP) chlorophyll fluorescence induction profiles. These revealed a distinct destabilisation of the QA- state in the PSII-I assembly intermediate and inactivated PSII samples related to an increased rate of direct and safe charge recombination. Furthermore, inactivation or removal of the OEC increases the binding affinity for plastoquinone analogues like DCBQ to the different PSII complexes. These results might indicate a mechanism that further contributes to the protection of PSII during biogenesis or repair.

The active site for water oxidation in Photosystem II (PSII) comprises a Mn4CaO5 cluster adjacent to a redox-active tyrosine residue (TyrZ). During the water-splitting process, the enzyme transitions through five sequential oxidation states (S0 to S4), with O2 evolution occurring during the S3TyrZ* to S0TyrZ transition. Chloride also plays a role in this mechanism. Using PSII from Thermosynechococcus vestitus, where Ca and Cl were replaced with Sr and Br to slow the S3TyrZ* to S0TyrZ + O2 transition (t1/2 [~] 5 ms at room temperature), it was observed that the recovery of a S0 state, defined as the state able to progress to S1, exhibits similar kinetics (t1/2 [~] 5 ms). This suggests that in CaCl-PSII, the reformation of the functional S0 state directly follows the S3TyrZ* to S0TyrZ + O2 transition, with no additional delay required for the insertion of a new substrate water molecule (O5) and associated protons.

Mitochondrial Complex III dysfunction is frequently associated with pathogenic variants in the MT-CYB gene, yet the functional consequences of many missense substitutions remain unresolved because they are classified as variants of uncertain significance (VUS). One such variant, m.14849T>C (p.Ser35Pro), has been reported in patients with multisystem mitochondrial phenotypes, including septo-optic dysplasia, cardiomyopathy, and exercise intolerance, although its structural impact on Cytochrome b function remains unclear. In this study, we employed 300 ns all-atom molecular dynamics simulations to assess structural and energetic consequences of the S35P substitution in the Cytochrome b subunit of human mitochondrial Complex III. The S35P variant did not induce global destabilization of the protein scaffold but instead promoted localized perturbations within the heme bL microenvironment. The mutation was associated with loss of a heme-proximal hydrogen-bonding network involving Ser35 and a decrease in electrostatic interaction energy between the protein matrix and the heme bL cofactor. Radial distribution function analysis further supported loosening of local packing around the prosthetic group. Consistent with these local changes, dynamics analyses indicated increased flexibility in distal transmembrane helices that form the heme-pocket scaffold and greater variability in the inter-heme Fe(bL)-Fe(bH) distance. Together, our findings suggest that S35P may exert functional effects by reorganizing the heme bL microenvironment rather than by inducing large-scale structural destabilization, underscoring the value of structure- and dynamics-based evaluation for mitochondrial VUS and suggesting a plausible mechanistic link to the pathophysiology of multisystem mitochondrial diseases.

Photosystem I is defined as plastocyanin-ferredoxin oxidoreductase. Taking advantage of genetic engineering, kinetic analyses and cryo-EM, our data provide novel mechanistic insights into binding and electron transfer between PSI and Pc. Structural data at 2.74 [A] resolution reveals strong hydrophobic interactions in the plant PSI-Pc ternary complex, leading to exclusion of water molecules from PsaA-PsaB / Pc interface once the PSI-Pc complex forms. Upon oxidation of Pc, a slight tilt of bound oxidized Pc allows water molecules to accommodate the space between Pc and PSI to drive Pc dissociation. Such a scenario is consistent with the six times larger dissociation constant of oxidized as compared to reduced Pc and mechanistically explains how this molecular machine optimized electron transfer for fast turnover. One Sentence SummaryGenetic engineering, kinetics and cryo-EM structural data reveal a mechanism in a major step of oxygenic photosynthesis

Anoxia halts oxidative phosphorylation (OXPHOS) causing an accumulation of reduced compounds in mitochondrial matrix which impedes dehydrogenases. By simultaneously measuring oxygen concentration, NADH autofluorescence, mitochondrial membrane potential and ubiquinone reduction extent in organello in real-time, we show that Complex I utilized endogenous quinones to oxidize NADH under acute anoxia. Untargeted or [U-13C]glutamate-targeted metabolomic analysis of matrix and effluxed metabolites extracted during anoxia in the presence or absence of site-specific inhibitors of the electron transfer system inferred that NAD+ regenerated by Complex I is reduced by the 2-oxoglutarate dehydrogenase complex yielding succinyl-CoA supporting mitochondrial substrate-level phosphorylation (mtSLP), releasing succinate. Yet, targeted metabolomic analysis using [U-13C]malate also revealed concomitant succinate dehydrogenase reversal during anoxia yielding succinate by reducing fumarate, albeit to a small extent. Our results highlight the importance of quinone availability to Complex I oxidizing NADH, thus maintaining glutamate catabolism and mtSLP in the absence of OXPHOS.

In the cyanobacterium Thermosynechococcus elongatus, there are three psbA genes coding for the Photosystem II (PSII) D1 subunit that interacts with most of the main cofactors involved in the electron transfers. Recently, the 3D crystal structures of both PsbA2-PSII and PsbA3-PSII have been solved [Nakajima et al., J. Biol. Chem. 298 (2022) 102668.]. It was proposed that the loss of one hydrogen bond of PheD1 due to the D1-Y147F exchange in PsbA2-PSII resulted in a more negative Em of PheD1 in PsbA2-PSII when compared to PsbA3-PSII. In addition, the loss of two water molecules in the Cl-1 channel was attributed to the D1-P173M substitution in PsbA2-PSII. This exchange, by narrowing the Cl-1 proton channel, could be at the origin of a slowing down of the proton release. Here, we have continued the characterization of PsbA2- PSII by measuring the thermoluminescence from the S2QA-/DCMU charge recombination and by measuring proton release kinetics using time-resolved absorption changes of the dye bromocresol purple. It was found that i) the Em of PheD1-*/PheD1 was decreased by [~] 30 mV in PsbA2-PSII when compared to PsbA3-PSII and ii) the kinetics of the proton release into the bulk was significantly slowed down in PsbA2-PSII in the S2TyrZ* to S3TyrZ and S3TyrZ* [->] (S3TyrZ*) transitions. This slowing down was partially reversed by the PsbA2/M173P mutation and induced by the PsbA3/P173M mutation thus confirming a role of the D1-173 residue in the egress of protons trough the Cl-1 channel.

Photosystem II uses water as the ultimate electron source of the photosynthetic electron transfer chain. Water is oxidized to dioxygen at the Oxygen Evolving Complex (OEC), a Mn4CaO5 inorganic core embedded in the lumenal side of PSII. Water-filled channels are thought to bring in substrate water molecules to the OEC, remove the substrate protons to the lumen, and may transport the product oxygen. Three water-filled channels, denoted large, narrow, and broad, that extend from the OEC towards the aqueous surface more than 15 [A] away are seen. However, the actual mechanisms of water supply to the OEC, the removal of protons to the lumen and diffusion of oxygen away from the OEC have yet to be established. Here, we combine Molecular Dynamics (MD), Multi Conformation Continuum Electrostatics (MCCE) and Network Analysis to compare and contrast the three potential proton transfer paths during the S1 to S2 transition of the OEC. Hydrogen bond network analysis shows that the three channels are highly interconnected with similar energetics for hydronium as calculated for all paths near the OEC. The channels diverge as they approach the lumen, with the water chain in the broad channel better interconnected that in the narrow and large channels, where disruptions in the network are observed at about 10 [A] from the OEC. In addition, the barrier for hydronium translocation is lower in the broad channel, suggesting that a proton from the OEC could access the paths near the OEC, and likely exit to the lumen via the broad channel, passing through PsbO.

Quinone-based electron bifurcation (EB) catalyzed by cytochrome bc1 (cytbc1) plays a critical role in maximizing efficiency of biological energy conversion. The canonical EB model (CEB), grounded in equilibrium redox potentials, dictates the order of EB steps with initial endergonic reduction of high-potential iron-sulfur cluster (2Fe2S) by quinol followed by exergonic reduction of low-potential heme bL (bL) by semiquinone (SQ). However, this concept falls short in explaining several experimental observations, including intermediate semiquinone spin-coupled to 2Fe2S (SQ-2Fe2Sred) and the absence of short-circuiting. Presented here DFT calculations on large cluster models of cytbc1, encompassing both 2Fe2S and bL, identified location of donor (HOMO) and acceptor (LUMO) orbitals along with the previously not considered microstates to reveal that EB is an emergent property of an integrated system of redox cofactors where transient charge separations dynamically modulate electron affinities. In this system, electron transfer initiates preferentially toward bL, indicating a departure from the conventional sequence proposed by CEB. Based on this finding, we introduce an EMBER (EMergent BL-first Electron Routing) model of EB and demonstrate that its assumptions are supported by electron paramagnetic resonance spectroscopy data. Unlike CEB, EMBER proposes a relatively flat energy profile for EB that accommodates stable SQ-2Fe2Sred and explains suppression of short-circuits without additional assumptions. It highlights the importance of state-dependent electrostatic interactions in shaping electron transfer pathways. In general, the concept of emergence inherent to EMBER offers a mechanistic framework applicable to a broad range of multi-cofactor redox enzymes beyond cytbc1.

Green organisms evolve O2 via photosynthesis and consume by respiration. Net O2 consumption only becomes dominant when photosynthesis is suppressed at night. Here, we show that green thylakoid membranes of Scots pine (Pinus sylvestris L) and Norway spruce (Picea abies) needles demonstrate strong O2 consumption even in the presence of light when extremely low temperatures coincide with high solar irradiation during early spring. This phenomenon deviates from the general finding that photosynthetic organisms evolve O2 upon illumination. By using different electron transport chain inhibitors, we showed that O2 consumption occurred around photosystem (PS) I and correlated with higher abundance of flavodiiron (Flv) A protein in ES thylakoid membranes. Furthermore, by measuring P700 absorption changes, we separated different alternative electron flow pathways and demonstrated that electron scavenging from the acceptor-side of PSI via O2 photoreduction is a major alternative pathway in ES. This photoprotection mechanism in vascular plants indicates that conifers have developed an adaptative evolution trajectory for growing in harsh environments.

DosT and DosS are heme-based kinases involved in sensing and signaling O2 tension in the microenvironment of Mycobacterium tuberculosis (Mtb). Under conditions of low O2, they activate >50 dormancy-related genes and play a pivotal role in the induction of dormancy and associated drug resistance during tuberculosis infection. In this work, we reexamine the O2 binding affinities of DosT and DosS to show that their equilibrium dissociation constants are 3.3{+/-}1 M and 0.46{+/-}0.08 M respectively, which are six to eight-fold stronger than what has been widely referred to in literature. Furthermore, stopped-flow kinetic studies reveal association and dissociation rate constants of 0.84 M-1s-1 and 2.8 s-1, respectively for DosT, and 7.2 M-1s-1 and 3.3 s-1, respectively for DosS. Remarkably, these tighter O2 binding constants correlate with distinct stages of hypoxia-induced non-replicating persistence in the Wayne model of Mtb. This knowledge opens doors to deconvoluting the intricate interplay between hypoxia adaptation stages and the signal transduction capabilities of these important heme-based O2 sensors.

This manuscript has been withdrawn by the authors due to disagreement with UCSF over data rights. This withdrawal decision is not related to the validity of the data presented in this study, and these authors understand that this work cannot be cited as reference for the project until the disagreement is resolved.

Most social insects are characterized by a wide disparity in life-history traits between individuals of the same species. Sterile workers live for months or years while queens may live for decades. Theories of ageing emphasise the importance of metabolism and oxidative stress in explaining longevity, with mitochondrial bioenergetics standing at the crossroads of energy and reactive oxygen species production. Studying mitochondrial functioning therefore takes on its full relevance in determining the nature of the mechanisms that explain the contrasting longevities between insect social castes. We addressed this question in an eusocial species, the black garden ant Lasius niger. We found that caste differences in mitochondrial bioenergetics and oxidative balance only partially match with predictions of the oxidative stress theory of ageing. Long-lived queens were characterized by a lower metabolic rate, lower mitochondrial density yet not necessarily lower levels of mitochondrial oxidative damages. Despite this, queens did not show reduced ATP content; rather, they even possessed a higher energy load in their mitochondria. Converging clues suggested better mitochondrial maintenance in queen ants, with enhanced dynamics of mitochondrial fission and fusion and a more marked expression of mitochondrial enzymes of the Krebs cycle. Overall, our data paves the way for studying deeper into how the rate of ATP production per mitochondria is related to the investment in mitochondrial and somatic cellular maintenance, and whether it has specifically been selected as a key mechanism in defining the still unexplained paradoxical longevity of the queen reproductive caste.

Calcium ion concentration modulates the function of pyruvate dehydrogenase, isocitrate dehydrogenase, and -ketoglutarate dehydrogenase. Previous studies have shown that despite its ability to affect the function of these dehydrogenases, [Ca2+] does not substantially alter mitochondrial ATP synthesis in vitro under physiological sub-strate conditions. We hypothesize that, rather than contributing to respiratory control, [Ca2+] governs fuel selection. Specifically, cardiac mitochondria are able to use different primary carbon substrates to synthesize ATP aerobically. To determine if and how [Ca2+] affects the relative use of carbohydrates versus fatty acids we measured oxygen consumption and tricarboxylic acid cycle intermediate concentrations in suspensions of cardiac mitochondria with different combinations of pyruvate and palmitoyl-L-carnitine in the media at various [Ca2+] and ADP infusion rates. Results reveal that when both fatty acid and carbohydrate substrates are available, fuel selection is sensitive to both calcium and ATP synthesis rate. When no Ca2+ is added under low ATP-demand conditions, {beta}-oxidation provides roughly half of acetyl-CoA for the citrate synthase reaction with the rest coming from the pyruvate dehydrogenase reaction. Under low demand conditions with increasing [Ca2+], the fuel utilization ratio shifts to increased fractional consumption of pyruvate, with 83{+/-}10% of acetyl-CoA derived from pyruvate at the highest [Ca2+] evaluated. With high ATP demand, the majority of acetyl-CoA is derived from pyruvate, regardless of the Ca2+ level. Our results suggest that changes in work rate alone are enough to effect a switch to carbohydrate use while in vivo the rate at which this switch happens may depend on mitochondrial calcium.\n\nKey PointsO_LIDespite its effects on activity of mitochondrial dehydrogenases, Ca2+ does not substantially alter mitochondrial ATP synthesis in vitro under physiological substrate conditions. Nor does is appear to play an important role in respiratory control in vivo in the myocardium.\nC_LIO_LIWe hypothesize that Ca2+ plays a role mediating the switch in fuel selection to increasing carbohydrate oxidation and decreasing fatty acid oxidation with increasing work rate.\nC_LIO_LITo determine if and how Ca2+ affects the relative use of carbohydrates versus fatty acids in vitro we measured oxygen consumption and TCA cycle intermediate concentrations in suspensions of purified rat ventricular mitochondria with carbohydrate, fatty acid, and mixed substrates at various [Ca2+] and ATP demand rates.\nC_LIO_LIOur results suggest that changes in work rate alone are enough to effect a switch to carbohydrate use in vitro while in vivo the rate at which this switch happens may depend on mitochondrial calcium.\nC_LI

Cytochrome bd is a distinctive family of terminal oxidases present in the respiratory chains of many prokaryotes. Despite its biological importance, the redox chemistry of these proteins remains poorly understood, largely due to the presence of two b-type hemes and one d-type heme. Here, we report the first computational study of inter-heme electron transfer in the cytochrome bd family. We performed 10 s of molecular dynamics simulations of E. coli cytochrome bd-I embedded in realistic membranes, combined with quantum chemical calculations to estimate the thermodynamic parameters of electron transfer from heme b595 to heme d within the framework of Marcus theory. We further identify the respective contributions of the hemes, protein scaffold, lipid bilayer, water, and counterions to the driving force and reorganization energy. The inter-heme electronic coupling was calculated using the Projected Orbital Diabatization (POD) method in a hybrid Quantum Mechanics/Molecular Mechanics scheme and rationalized through electron transfer pathway analysis. This study provides fundamental insights into how electron transfer steps are orchestrated in the catalytic cycle of E. coli cytochrome bd-I. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/673948v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@14a29c8org.highwire.dtl.DTLVardef@1fd1c98org.highwire.dtl.DTLVardef@6053aaorg.highwire.dtl.DTLVardef@14ec40_HPS_FORMAT_FIGEXP M_FIG C_FIG