Abstract
Phenazines are a class of redox-active molecules produced by diverse bacteria and archaea. Many of the biological functions of phenazines, such as mediating signaling, iron acquisition, and redox homeostasis, derive from their redox activity. Although prior studies have focused on extracellular phenazine oxidation by oxygen and iron, here we report a search for reductants and catalysts of intracellular phenazine reduction in Pseudomonas aeruginosa Enzymatic assays in cell-free lysate, together with crude fractionation and chemical inhibition, indicate that P. aeruginosa contains multiple enzymes that catalyze the reduction of the endogenous phenazines pyocyanin and phenazine-1-carboxylic acid in both cytosolic and membrane fractions. We used chemical inhibitors to target general enzyme classes and found that an inhibitor of flavoproteins and heme-containing proteins, diphenyleneiodonium, effectively inhibited phenazine reduction in vitro, suggesting that most phenazine reduction derives from these enzymes. Using natively purified proteins, we demonstrate that the pyruvate and α-ketoglutarate dehydrogenase complexes directly catalyze phenazine reduction with pyruvate or α-ketoglutarate as electron donors. Both complexes transfer electrons to phenazines through the common subunit dihydrolipoamide dehydrogenase, a flavoprotein encoded by the gene lpdG Although we were unable to co-crystallize LpdG with an endogenous phenazine, we report its X-ray crystal structure in the apo-form (refined to 1.35 Å), bound to NAD+ (1.45 Å), and bound to NADH (1.79 Å). In contrast to the notion that phenazines support intracellular redox homeostasis by oxidizing NADH, our work suggests that phenazines may substitute for NAD+ in LpdG and other enzymes, achieving the same end by a different mechanism.
Highlights
Phenazines are a class of redox-active molecules produced by diverse bacteria and archaea
Phenazine Reduction Is Catalyzed by Cell Lysate—We first asked whether P. aeruginosa produces enzymes that catalyze phenazine reduction
Because phenazines alter the metabolic flux of P. aeruginosa [7, 20], we reasoned that intermediates of central metabolism might serve as electron donors, and so we tested pyruvate, citrate, isocitrate, ␣-ketoglutarate, succinate, and malate
Summary
Phenazines are a class of redox-active molecules produced by diverse bacteria and archaea. We showed that phenazines, by acting as electron shuttles, expand the habitable zone of colony biofilms into the anoxic portion [8] These findings paint a nuanced view where phenazines are multifunctional molecules, in some contexts possessing antibiotic activity and in others supporting redox homeostasis or iron acquisition. This diverse array of structurally similar molecules suggests particular phenazines might be tuned for certain functions or properties In support of this interpretation, the endogenous P. aeruginosa phenazines have different pH-dependent solubilities, toxicities, and reactivities toward oxygen and iron [16, 27]. Despite their structural diversity, the key biological functions of phenazines, be it promoting anaerobic energy generation or facilitating iron acquisition, derive from their redox activity. Our data indicate that under energy-limited anoxic conditions, phenazine substitution for NADϩ in these enzymes might contribute to the maintenance of intracellular redox homeostasis
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