In Saccharomyces cerevisiae, cytosolic NADH generated during biomass production must be efficiently reoxidized to sustain metabolic flux. Under aerobic conditions, this process is primarily mediated by the oxidative phosphorylation and specifically via external NADH dehydrogenases (Nde1p/Nde2p) and the glycerol-3-phosphate (G3P) shuttle. Although these pathways perform equivalent redox functions, their relative contributions vary with physiological conditions, indicating the existence of regulatory mechanisms governing electron partitioning.Here, we combined experimental measurements and kinetic modeling to investigate the mechanisms underlying electron competition between NADH and G3P oxidation in isolated yeast mitochondria. Using wild-type and Δnde1 strains, we determined the kinetic parameters of the relevant dehydrogenases and analyzed their dependence on the quinone redox state. A mathematical model incorporating these parameters reproduced the experimentally observed preferential utilization of NADH in wild-type mitochondria as well as the simultaneous oxidation of NADH and G3P in the Δnde1 mutant.Our results demonstrate that, under conditions where the downstream respiratory chain is rate-limiting, electron partitioning is governed by the ratio of catalytic efficiencies (Vmax/K0.5) of NADH and G3P dehydrogenases with respect to the quinone redox ratio (Q/QH₂).These findings identify a simple kinetic principle underlying substrate competition in mitochondrial electron transport and provide a quantitative framework for understanding metabolic flux distribution in eukaryotic cells.
In eukaryotic light-harvesting complexes, the protein host tunes chlorophyll-a (Chl a) site energies through a number of mechanisms (e.g. hydrogen bonding; excitonic/charge-transfer (CT) mixing). However, whether protein-imposed macrocycle distortions also contribute to fine-tuning this energy remains unclear. We have investigated three Chl a-only algal antennas - VCP (from Nannochloropsis oceanica), rVCP (Trachydiscus minutus), and CLH (Chromera velia). These complexes show distinct red-shifted absorption features, which at 77 K range from 670 nm up to an extremely red-shifted band at 700 nm (rVCP). By analysis of marker bands in excitation-selective resonance Raman spectra, we have determined the contribution of (i) hydrogen bonding to the keto group (1630–1700 cm−1 region), and/or (ii) macrocycle distortion (bands near 1555 and 1612 cm−1) to these red-shifts. Finally, the absence of vibrational features in fluorescence line-narrowing measurements for the reddest rVCP band is consistent with strong excitonic coupling, which we suggest is at the origin of this large red shift. Our results indicate that tuning of the functional properties of Chl a antennas may occur via distortion of the macrocycle.
Cyanobacterial NDH-1 complexes use a conserved Type-I energy-transducing core for respiratory electron transfer, photosystem I cyclic electron flow, and, in specialized isoforms, active CO2 uptake. This review examines how the common structural and energetic principles of respiratory Complex I may be adapted to these distinct physiological functions. Building on current mechanistic models, this review frames quinone reduction as producing a distributed, metastable "tense state" in the membrane arm. This state is represented not by a single strained conformation, but by coupled changes in proton affinity, ion-pair geometry, hydration, and local electrostatics. This framework is then applied to the cyanobacterial CO2-uptake complexes NDH-13 and NDH-14, in which the distal NdhF3/NdhF4-CupA/B module appears to repurpose part of the canonical proton-coupling machinery for Zn-dependent CO2 hydration. Bioenergetic estimates indicate that ferredoxin-dependent plastoquinone reduction could, in principle, support three-proton translocation together with CO2 hydration and bicarbonate accumulation. The model connects Complex I energy transduction pathways to cyanobacterial CO2 hydration chemistry and identifies testable predictions regarding coupling at the Zn-site to distant plastoquinone reduction and proton transfer.
Water oxidation in photosynthesis occurs through a four-electron redox reaction that is catalyzed by the oxygen-evolving complex (OEC) in Photosystem II (PS II), that consists of the Mn4CaO5 cluster. Among the four stable intermediates in the water oxidation reaction, it has been reported that the paramagnetic S2 state with the formal oxidation of Mn(III)Mn(IV)3 has multiple spin states, that include high spin (HS, 5/2, g≥4.1) and low spin (LS, 1/2, g = 2) forms. Previous computational studies proposed that these two forms differ in their structure and the high spin form may represent an intermediate form of the OEC during the S2 to S3 transition, the step in which the first substrate water binds to the last open coordination site of the cluster. In the current study, we examined the pH-induced HS-S2 state of T. vestitus PS II with MnK edge extended X-ray absorption fine structure (EXAFS) and X-ray absorption near edge spectroscopy (XANES). Small, but notable differences were observed in the EXAFS and XANES between the HS and LS S2 states. EXAFS data and the fits indicate that the basic geometry of the cluster remains the same between these two states. Therefore, we hypothesize that the difference between the HS and LS S2 state may be due to chemical differences such as different protonation states of the oxo and/or water ligands, or its surroundings. We also examined the detailed oxygen positions of the S2 states in the XFEL-based room temperature crystal structures, and confirmed that at neutral pH at room temperature, the structure is consistent with that of the LS-S2 model with open cubane-like geometry.
