Biology subjects

Greene, B. L.

Publications and source records attributed to Greene, B. L..

4 recordsLinked to original sources

bioRxiv2025

Measurement and Control of Crossed Potentials in a Flavoprotein

Flavoproteins are versatile redox-active biomolecules enabling a multitude of metabolic processes. Their versatility stems from the tunability of the flavin cofactors one- and two-electron reduction potentials via interactions with the protein scaffold, which have dramatic influence on reactivity. Although several mechanisms have been proposed to explain how the flavin-binding pocket modulates redox thermodynamics, few have been validated through quantitative experiments. In this study, we investigate how the flavin N5 environment influences the redox properties of the flavin mononucleotide cofactor in the "improved" light-oxygen-voltage (iLOV) sensing protein using site-directed mutagenesis, redox titrations, and hybrid quantum mechanical molecular mechanical (QM/MM) methods combined with classical alchemical free energy simulations. Mutating the residue Q103, which interacts with the flavin N5 and O4' atoms in the X-ray crystallographic structure, exerts a modest < 35 mV effect on the overall two-electron reduction potential, but significantly alters the potential separation of the two one-electron couples (potential crossing) by up to 168 mV. QM/MM and free energy calculations reveal that water penetration into the flavin binding pocket near N5 and O4' largely explains the trend in reduction potentials among the mutants. The results suggest a molecular mechanism of flavin tuning in which hydrogen bonding to the neutral semiquinone, either directly by the side-chain or a protein-penetrating water, contributes significantly to the potential crossing. These findings establish quantitative experimental benchmarks for theoretical models and advance a molecular mechanism for redox tuning in flavoproteins.

biochemistry↗

bioRxiv2025

Phosphite Production by Streptomyces viridochromogenes

The industrial production of phosphochemicals is highly energy-intensive, involving the reduction of the phosphate mineral apatite to white phosphorus, a toxic and reactive intermediate with significant environmental risk. Phosphite, an activated form of phosphorus, is a potential alternative substrate for phosphochemical synthesis, yet direct reduction of phosphate to phosphite remains challenging. While environmental and microbial studies have suggested biochemical pathways for reducing phosphate to phosphite, these pathways have not been conclusively demonstrated in axenic culture. In this study, we characterize phosphite production by Streptomyces viridochromogenes and demonstrate that phosphite is an abiological product of phosphonoformyl-CMP decomposition, an intermediate in the biosynthesis of the herbicide phosphinothricin. The phosphonoformyl-CMP intermediate yields an "activated" phosphonoformate for decarboxylation, producing phosphite at biological temperatures and pH following phosphoanhydride hydrolysis. Using S. viridochromogenes spent media, we demonstrate a hybrid biotic-abiotic synthesis of the metal chelator aminotris(methylenephosphonate), illustrating a potential synthetic route to phosphochemicals from biogenic phosphite.

biochemistry↗

bioRxiv2025

Nitric Oxide Inhibition of Glycyl Radical Enzymes and Their Activases

Innate immune response cells produce high concentrations of the free radical nitric oxide (NO) in response to pathogen infection. The antimicrobial properties of NO include non-specific damage to essential biomolecules and specific inactivation of enzymes central to aerobic metabolism. However, the molecular targets of NO in anaerobic metabolism are less understood. Here, we demonstrate that the Escherichia coli glycyl radical enzyme pyruvate formate lyase (PFL), which catalyzes the anaerobic metabolism of pyruvate, is irreversibly inhibited by NO. Using electron paramagnetic resonance and site-directed mutagenesis we show that NO destroys the glycyl radical of PFL. The activation of PFL by its cognate radical S-adenosyl-L-methionine-dependent activating enzyme (PFL-AE) is also inhibited by NO, resulting in the conversion of the essential iron-sulfur cluster to dinitrosyl iron complexes. Whole-cell EPR and metabolic flux analyses of anaerobically growing Escherichia coli show that PFL and PFL-AE are inhibited by physiologically relevant levels of NO in bacterial cell cultures, resulting in diminished growth and a metabolic shift to lactate fermentation. The class III ribonucleotide reductase (RNR) glycyl radical enzyme and its corresponding RNR-AE are also inhibited by NO in a mechanism analogous to those observed in PFL and PFL-AE, which likely contributes to the bacteriostatic effect of NO. Based on the similarities in reactivity of the PFL/RNR and PFL-AE/RNR-AE enzymes with NO, the mechanism of inactivation by NO appears to be general to the respective enzyme classes. The results implicate an immunological role of NO in inhibiting glycyl radical enzyme chemistry in the gut.

biochemistry↗

bioRxiv2024

Non-Canonical Cytochrome P450 Enzymes in Nature

Cytochrome P450s (CYPs) are a superfamily of thiolate-ligated heme metalloenzymes principally responsible for the hydroxylation of unactivated C-H bonds. The lower-axial cysteine is an obligatory and universally conserved residue for the CYP enzyme class. Herein, we challenge this paradigm by systematically identifying non-canonical CYPs (ncCYPs) that do not harbor a cysteine ligand. Our bioinformatic search reveals 20 distinct ncCYP families with diverse ligands encoded in microbial genomes. We characterize a native serine-ligated CYP with a high-spin ferric resting state. Its crystal structure clearly shows a typical CYP fold and a serine alkoxide as a lower axial heme ligand. In addition, we report the discovery and characterization of the first native selenocysteine-ligated CYP in nature. Our findings radically expand the CYP metalloenzyme family.

biochemistry↗