About

J. Martin Bollinger is a Professor of Chemistry and Biochemistry and Molecular Biology at The Pennsylvania State University. His research focuses on the mechanisms of metalloenzymes and metallofactor assembly. He is affiliated with the Molecular, Cellular, and Integrative Biosciences program and is involved in various institutes and centers related to bioremediation, infectious disease, industrial biotechnology, and neuroscience. His work includes studying heme oxygenase-like metalloenzymes, cyanobacterial halogenases, radical-initiating metallocofactors, and iron-dependent oxygenases, contributing to the understanding of enzyme mechanisms and biosynthesis pathways.

Research topics

• Chemistry
• Biochemistry
• Stereochemistry
• Biology
• Internal medicine
• Photochemistry
• Medicinal chemistry
• Organic chemistry
• Virology

Selected publications

• A Structurally Divergent Class Ia Ribonucleotide Reductase from a Tick-Borne Pathogen

  Biochemistry · 2025-08-28

  articleOpen accessCorresponding

  Ribonucleotide reductases (RNRs) generate 2′-deoxynucleotides for DNA biosynthesis, a reaction essential to all life. Class I RNRs have two subunits, α and β. α binds and reduces the substrate, whereas β oxidizes one of the cysteines in α to a C3′–H-bond-cleaving thiyl radical to begin the reaction. The α-Cys oxidant in β is variously a tyrosyl radical (Y•) generated by a diiron or dimanganese cluster, a high-valent dimetal cluster [Mn(IV)/Fe(III) or Mn2(IV/III)], or a dihydroxylphenylalanine (DOPA) radical that operates without need of a transition metal. The metal (in)dependence of the Cys oxidant in β correlates loosely with sequence-similarity groupings. We show here that Francisella hispaniensis (Fh) β, which lies within an uncharacterized sequence cluster that contains orthologs from multiple human pathogens, harbors a Fe2(III/III)/Y• cofactor, as in class Ia RNRs from eukaryotes and Escherichia coli. Fh β has several unusual structural features that may reflect adaptation to the bacterium’s environment(s). In its apo form, an unwound helix everts a metal ligand toward solvent, and the radical-harboring Y points away from the diiron cluster. An additional aromatic residue (W194), conserved within the sequence cluster, is found close to the universally conserved W37, which is thought to mediate α-Cys oxidation in all class I enzymes. The Y• in resting β is remarkably resistant to reduction by hydroxyurea but becomes 8000 times more sensitive when β is engaged in turnover with α. These structural and functional distinctions could be counter measures against host redox defenses that would target the pathogen’s RNR and its cofactor.

  PublisherOA PDFDOI

• Biochemical Studies of a Cyanobacterial Halogenase Support the Involvement of a Dimetal Cofactor

  Biochemistry · 2025-04-29 · 2 citations

  articleOpen accessCorresponding

  Halogenation is a prominent transformation in natural product biosynthesis, with over 5000 halogenated natural products known to date. Biosynthetic pathways accomplish the synthetic challenge of selective halogenation, especially at unactivated sp3 carbon centers, using halogenase enzymes. The halogenase CylC, discovered as part of the cylindrocyclophane (cyl) biosynthetic pathway, performs a highly selective chlorination reaction on an unactivated sp3 carbon center and is proposed to use a dimetal cofactor. Putative dimetal halogenases are widely distributed across cyanobacterial biosynthetic pathways. However, rigorous in vitro biochemical and structural characterization of these enzymes has been challenging. Here, we report additional bioinformatic analyses of putative dimetal halogenases and the biochemical characterization of a newly identified CylC homologue. Site-directed mutagenesis identifies highly conserved putative metal-binding residues, and Mössbauer spectroscopy provides direct evidence for the presence of a diiron cofactor in these halogenases. These insights suggest mechanistic parallels between diiron and mononuclear nonheme iron halogenases, with the potential to guide further characterization and engineering of this unique subfamily of metalloenzymes.

  PublisherDOI

• [Withdrawn] Hydralazine Inhibits Cysteamine Dioxygenase to Treat Preeclampsia and Senesce in Glioblastoma

  Qeios · 2025-01-17

  preprintOpen access

  This manuscript has been withdrawn.

  PublisherOA PDFDOI

• Iron-sulfur clusters in SARS-CoV-2 exoribonuclease and methyltransferase complexes: relevance for viral genome proofreading and capping

  Nature Communications · 2025-08-15 · 3 citations

  articleOpen access

  Abstract Coronaviruses rely on a multifunctional replication-transcription complex to ensure genome fidelity and support viral propagation. Within this complex, the nsp14-nsp10 heterodimer possesses 3’−5’ exoribonuclease (ExoN) activity, while nsp14 alone functions as an N7-methyltransferase and the nsp16/nsp10 complex completes viral RNA capping via its 2′-O-methyltransferase. Here, we report that nsp14 and nsp10 ligate [Fe 4 S 4 ] clusters when purified anoxically, in sites previously modeled as zinc centers. Quantum mechanics/molecular mechanics simulations revealed distinct reduction potentials for these iron-sulfur (Fe-S) clusters, and redox titrations demonstrated that changes in oxidation state modulate RNA binding by nsp14 and the nsp10/nsp16 complex. Functionally, Fe-S clusters enhance the methyltransferase activities of nsp14 and nsp10/nsp16, while leaving the ExoN activity unaffected. These findings uncover a redox-regulated role for Fe-S clusters in SARS-CoV-2 RNA processing and suggest that the viral core enzymatic functions may be modulated by the redox state of their Fe-S cofactors.

  PublisherOA PDFDOI

• Structural Elucidation of the Reduced Mn(III)/Fe(III) Intermediate of the Radical-Initiating Metallocofactor in <i>Chlamydia trachomatis</i> Ribonucleotide Reductase

  Biochemistry · 2025-02-17 · 3 citations

  articleOpen access

  Ribonucleotide reductases (RNRs) are the sole de novo source of deoxyribonucleotides for DNA synthesis and repair across all organisms and carry out their reaction via a radical mechanism. RNR from Chlamydia trachomatis generates its turnover-initiating cysteinyl radical by long-range reduction of a Mn(IV)/Fe(III) cofactor, producing a Mn(III)/Fe(III) intermediate. Herein, we characterize the protonation states of the inorganic ligands in this reduced state using advanced pulse electron paramagnetic resonance (EPR) spectroscopy and 2H-isotope labeling. A strongly coupled deuteron is observed by hyperfine sublevel correlation (HYSCORE) spectroscopy experiments and indicates the presence of a bridging hydroxo ligand. Isotope-dependent EPR line broadening analysis and the magnitude of the estimated Mn–Fe exchange coupling constant together suggest a μ-oxo/μ-hydroxo core. Two distinct signals detected in electron–nuclear double resonance (ENDOR) spectra are attributable to less strongly coupled hydrons of a terminal water ligand to Mn(III). Together, these experiments imply that the reduced cofactor has a mixed μ-oxo/μ-hydroxo core with a terminal water ligand on Mn(III). This structural assignment sheds light generally on the reactivity of Mn/Fe heterobimetallic sites and, more specifically, on the proton-coupling in the electron transfer that initiates ribonucleotide reduction in this subclass of RNRs.

  PublisherDOI

• Azetidine amino acid biosynthesis by non-haem iron-dependent enzymes

  Nature Chemistry · 2025-10-21 · 5 citations

  articleOpen access

  Abstract Azetidine, a four-membered aza-cycle, is a crucial structure in many bioactive compounds and drugs. However, their biosynthesis is frequently enigmatic. Here we report the mechanism of azetidine amino acid (polyoximic acid) biosynthesis in the polyoxin antifungal pathway. Genetic, enzymological and structural experiments revealed that PolF is a member of haem-oxygenase-like dimetal oxidase and/or oxygenase (HDO) superfamily, and this enzyme alone is sufficient for the transformation of l -isoleucine ( l -Ile) and l -valine to their azetidine derivatives via a 3,4-desaturated intermediate. Mechanistic studies of PolF suggested that a μ-peroxo-Fe(III) 2 intermediate is directly responsible for the unactivated C–H bond cleavage, and the post-H-abstraction reactions, including the C–N bond formation, probably proceed through radical mechanisms. We also found that PolE, a member of the DUF6421 family, is an Fe and pterin-dependent oxidase that catalyses the desaturation of l -Ile, assisting PolF by increasing the flux of l -Ile desaturation. The results provide important insights into azetidine biosynthesis and the catalytic mechanisms of HDO enzymes in general.

  PublisherOA PDFDOI

• Experimental and Computational Analysis of the Inability of an Iron(II)- and 2-Oxoglutarate-Dependent Aliphatic Halogenase to Mediate Fluorination

  ChemRxiv · 2025-11-19 · 1 citations

  articleOpen accessSenior author

  Incorporation of fluorine into pharmaceuticals, agrochemicals, and molecular-imaging agents is of growing importance. Multiple synthetic fluorination methods have recently emerged, and metalloenzymes that are potentially capable of even C(sp3)–H fluorination have been reported. Nevertheless, direct, regioselective fluorination of aliphatic carbon centers remains an unsolved problem. Here, we show for the iron(II) and 2-oxoglutarate-dependent (Fe/2OG) L-Lysine 4-chlorinase, BesD, which can be envisaged to support C(sp3)–H fluorination by the direct cognate of its native chlorination mechanism, that the enzyme can (1) coordinate F– at its Fe(II) cofactor, (2) activate O2 to form a cis-FeIV(O)(F) (fluoroferryl) intermediate, and (3) use the intermediate to abstract hydrogen from its substrate. In what would be the key final step, fluorine (F•) transfer to the substrate radical is unable to compete with the hydroxyl-radical (HO•) "rebound" step characteristic of related hydroxylases. Electron paramagnetic resonance (EPR) and X-ray absorption spectroscopic data establish that fluorine remains bonded to the cofactor through steps 1-3 and therefore available for transfer to the substrate radical. QM/MM calculations suggest that the F•-coupling step is associated with an activation barrier considerably higher than that of HO• rebound, consistent with the observed outcome. The findings experimentally verify prior proposals that the impediment to C(sp3)–H fluorination by the canonical mechanism of an Fe/2OG halogenase lies in the final radical-coupling step and set the stage for exploration of whether a potentially surmountable geometric barrier or an insurmountable electronic one is primarily responsible.

  PublisherDOI

• Dynamic metal coordination controls chemoselectivity in a radical halogenase

  Nature Chemical Biology · 2025-11-21 · 4 citations

  articleOpen access
  PublisherOA PDFDOI

• A ferritin-like diiron oxygenase BioE initiates bacterial biotin synthesis, a promising antivirulence target

  Proceedings of the National Academy of Sciences · 2025-12-04 · 4 citations

  articleOpen accessCorresponding

  Biotin is an essential enzyme cofactor for intermediary metabolism, and its importance is reflected by the multiplicity of bacterial pathways to its universal precursor, pimelic acid. Here, we report identification of a fourth pimeloyl pathway in the rare but clinically important pathogens Elizabethkingia and Chryseobacterium . This pathway is encoded by two associated structural genes, bioE and bioL . BioE is a ferritin-like nonheme diiron oxygenase that oxidatively cleaves saturated C n (n = 14, 16, 18) fatty acyl coenzyme A (CoA) or acyl carrier protein (ACP) substrates to pimeloyl-CoA/ACP and the free C n-7 acid. The catalytic activity was demonstrated by both in vitro enzymatic assays and the capacity of the bioE gene to complement the genetic defect of an Escherichia coli biotin indicator strain that cannot produce the pimeloyl precursor. BioL, an unusual MocR-type bifunctional transcription factor, negatively regulates bioE expression in response to binding of the downstream intermediate 7-keto-8-aminopelargonic acid. Disruption of bioE in Elizabethkingia meningoseptica and Chryseobacterium indologenes makes them auxotrophic for biotin, impairs biofilm formation, and attenuates bacterial infectivity. Taken together, our findings expand enzymatic diversity of biotin biosynthesis and suggest that selective inhibition of this BioE pathway could provide a therapeutic strategy against recalcitrant nosocomial infections caused by these multidrug-resistant pathogens.

  PublisherOA PDFDOI

• Heme Oxygenase–Like Metalloenzymes

  Annual Review of Biochemistry · 2025-03-27 · 13 citations

  reviewOpen access

  Heme oxygenase (HO)-like metalloenzymes are an emerging protein superfamily diverse in reaction outcome and mechanism. Found primarily in bacterial biosynthetic pathways, members conserve a flexible protein scaffold shared with the heme catabolic enzyme, HO, and a set of metal-binding residues. Most HO-like metalloenzymes assemble a diiron cluster, although manganese-iron and mononuclear iron cofactors can also be accommodated. In the canonical HO-like diiron oxygenases/oxidases (HDOs), an Fe 2 (II/II) complex reacts with O 2 to form a peroxo-Fe 2 (III/III) intermediate ( P ), common to all HDOs studied to date. The HO-like scaffold confers both distinctive metal-binding properties, allowing for dynamic cofactor assembly and disassembly, and unusual reactivity to its associated metallocofactor. These features may prove to be important in HDO-mediated catalysis of the fragmentation and rearrangement reactions that remain unprecedented among other dinuclear iron enzymes. Much of the sequence space in the HO-like metalloenzyme superfamily remains unexplored, offering exciting opportunities for the discovery of new mechanisms and reactivities.

  PublisherDOI

Recent grants

• NIH Grant R01DK074641

  NIH · $1.1M · 2011

• Mechanisms and Reprogramming of Iron/2-Oxoglutarate Desaturases and Oxacyclases

  NIH · $1.0M · 2016–2021

• NIH Grant R01GM055365

  NIH · $3.7M · 2013

• Mechanisms of oxacycle- and olefin-installing iron/2-(oxo)glutarate oxygenases

  NIH · $1.8M · 2015–2020

• Mechanism of Taurine: Alpha-Ketoglutarate Dioxygenase

  NIH · $2.7M · 2004–2018

Frequent coauthors

• Carsten Krebs

  Pennsylvania State University

  211 shared

• Boi Hanh Huynh

  64 shared

• Eric W. Barr

  University of Pennsylvania

  59 shared

• Lana Saleh

  55 shared

• Christopher T. Walsh

  Met Office

  52 shared

• Jeffrey Baldwin

  The University of Texas Southwestern Medical Center

  46 shared

• Edward I. Solomon

  SLAC National Accelerator Laboratory

  44 shared

• Brenda A. Ley

  Pennsylvania State University

  43 shared