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Energy homeostasis is a conserved process: Evidence from Paracoccus denitrificans’ response to acute changes in energy demand

ABSTRACT: Paracoccus denitrificans is a model organism for the study of oxidative phosphorylation. We demonstrate a very high respiratory capacity compared to mitochondria when normalizing to cytochrome aa3 content even in the absence of alternative terminal oxidases. To gain insight into conserved mechanisms of energy homeostasis, we characterized the metabolic response to K+ reintroduction. A rapid 3-4-fold increase in respiration occurred before substantial cellular K+ accumulation followed by a sustained increase of up to 6-fold that persisted after net K+ uptake stopped. Proton motive force (Δp) was slightly higher upon addition of K+ with ΔpH increasing and compensating for membrane potential (ΔΨ) depolarization. Blocking the F0F1-ATP synthase (Complex V) with venturicidin revealed that the initial K+-dependent respiratory activation was primarily due to K+ influx. However, the ability to sustain an increased respiration rate was partially dependent on Complex V activity. The 6-fold stimulation of respiration by K+ resulted in a small net reduction of most cytochromes, different from the pattern observed with chemical uncoupling and consistent with balanced input and utilization of reducing equivalents. Metabolomics showed increases in glycolytic and TCA cycle intermediates together with a decrease in basic amino acids, suggesting an increased nitrogen mobilization upon K+ replenishment. ATP and GTP concentrations increased after K+ addition, indicating a net increase in cellular potential energy. Thus, K+ stimulates energy generation and utilization resulting in an almost constant Δp and increased high-energy phosphates during large acute and steady state changes in respiration. The specific energy consuming processes and signaling events associated with this simultaneous activation of work and metabolism in P. denitrificans remain unknown. Nevertheless, this homeostatic behavior is very similar to that observed in mitochondria in tissues when cellular energy requirements increase. We conclude that the regulation of energy generation and utilization to maintain homeostasis is conserved across the prokaryote/eukaryote boundary.

SUBMITTER: Covian R

PROVIDER: S-EPMC8575270 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Variant nomenclature: the variants were made in the NorB subunit if not indicated by the superscript c, which are variants in the NorC subunit (e.g. E122A = exchange of Glu-122 in NorB for an Ala, E71cD; exchange of Glu-71 in NorC for an Asp). Bacterial NO reductases (NORs) are integral membrane proteins from the heme-copper oxidase superfamily. Most heme-copper oxidases are proton-pumping enzymes that reduce O2 as the last step in the respiratory chain. With electrons from cytochrome c, NO reductase (cNOR) from Paracoccus (P.) denitrificans reduces NO to N2O via the following reaction: 2NO+2e-+2H+?N2O+H2O. Although this reaction is as exergonic as O2-reduction, cNOR does not contribute to the electrochemical gradient over the membrane. This means that cNOR does not pump protons and that the protons needed for the reaction are taken from the periplasmic side of the membrane (since the electrons are donated from this side). We previously showed that the P. denitrificans cNOR uses a single defined proton pathway with residues Glu-58 and Lys-54 from the NorC subunit at the entrance. Here we further strengthened the evidence in support of this pathway. Our further aim was to define the continuation of the pathway and the immediate proton donor for the active site. To this end, we investigated the region around the calcium-binding site and both propionates of heme b3 by site directed mutagenesis. Changing single amino acids in these areas often had severe effects on cNOR function, with many variants having a perturbed active site, making detailed analysis of proton transfer properties difficult. Our data does however indicate that the calcium ligation sphere and the region around the heme b3 propionates are important for proton transfer and presumably contain the proton donor. The possible evolutionary link between the area for the immediate donor in cNOR and the proton loading site (PLS) for pumped protons in oxygen-reducing heme-copper oxidases is discussed.

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Energy homeostasis is a conserved process: Evidence from Paracoccus denitrificans’ response to acute changes in energy demand

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