Subcellular-scale directed green synthesis of nanoparticles: First insight into protein-level regulatory mechanisms of electron donors on palladium nanoparticle biosynthesis pathways in Bacillus megaterium Y-4

Microbially synthesized palladium nanoparticles (bio-Pd 0 ) hold great promise for catalysis, but the catalytic activity of bio-Pd 0 relies heavily on its subcellular localization. Electron donors could affect enzymatic reduction efficiency of Pd(II) and control the subcellular location of bio-Pd 0 , yet the underlying mechanisms were unclear. Here, the regulatory mechanism of electron donors on Pd(II) reduction pathways in the non-model bacterium B. megaterium Y-4 was comprehensively investigated by multiple methodologies including cell fractionation, DPV/CV, XPS, FTIR, TEM, proteomics. Key findings revealed that membrane-bound hydrogenases drove periplasmic Pd 0 synthesis via biohydrogen, while the new one-electron extracellular transfer (EET) channel consisting of membrane-attached cytochromes ( ccsB ) and multi-heme cytochromes-bound flavin mediated the extracellular Pd(II) reduction. Notably, electron donors could regulate Pd(II) reduction routes, thereby altering the subcellular distribution of bio-Pd 0 . In lactate-added systems, the higher expression of these proteins related to EET processes (NADH dehydrogenase, membrane-attached cytochrome c, heme-based electron transfer proteins and flavodoxin) promoted extracellular Pd 0 formation (65.4 %). Whereas, in formate-added systems, hydrogenase-driven periplasmic Pd 0 synthesis (64.0 %) was facilitated due to more active formate dehydrogenase, hydrogenase and NADPH-reducing hydrogenase. Moreover, synergistical metabolism of formate and lactate balanced both pathways and maximized the apparent Pd(II) reduction efficiency (98.13 %). Proteomic analysis confirmed formate-specific energy rewiring and compensatory energy via Na⁺-translocating Rnf complex and V-ATPase overexpression. These findings significantly advance our understanding of microbial metal reduction by delineating a true dual-pathway strategy in a Gram-positive bacterium, highlighting the mechanistic diversity beyond model Gram-negative systems and offering new insights for harnessing non-model organisms in biocatalysis and nanoparticle synthesis.