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.