Intracellular Proton Enrichment Drives Unified Dual-Cofactor Regeneration for Biohybrid CO2 Fixation.

Photocatalytic biohybrid systems, which interface photosensitizers with whole-cell biocatalysts, represent a platform for sustainable solar-to-chemical CO2 conversion. However, their efficiency is fundamentally constrained by kinetic mismatch between sluggish transmembrane electron injection and uncoupled proton flux required for bioenergetic transduction. This uncoupling restricts the regeneration of adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH), the dual cofactors essential for driving carbon fixation pathways. Here, we introduce a proton enrichment strategy utilizing protonated manganese-doped carbon dots (HMnCDs) that function simultaneously as reversible proton buffers and photoelectron donors. HMnCDs localize to the periplasm of Cupriavidus necator H16, where their labile protons reinforce the transmembrane proton gradient driving ATP synthase. Concurrently, cytoplasmic HMnCDs facilitate proton-coupled electron transfer, accelerating NADH photoregeneration. The concerted management of proton and electron fluxes decouples ATP generation from respiratory NADH oxidation, creating a synergistic cofactor supply even under electron transport chain inhibition. Consequently, the biohybrid achieves light-driven autotrophic growth and poly(3-hydroxybutyrate) biosynthesis from CO2, attaining a record quantum efficiency of 20.8%. Multi-omics analyses reveal global metabolic reprogramming, including upregulated carbon fixation pathways and adaptive modulation of energy homeostasis under the proton-enriched microenvironment. This work establishes proton enrichment as a generalizable design principle for coupling photochemistry with cellular bioenergetics.