About

Carsten Krebs is a Distinguished Professor of Chemistry and Biochemistry and Molecular Biology at Pennsylvania State University, serving also as the Associate Department Head for Chemistry Climate and Diversity. His research focuses on bioinorganic chemistry, specifically spectroscopic and kinetic studies on the mechanisms of iron-containing enzymes. His interdisciplinary program aims to combine biochemical, kinetic, and spectroscopic methods, primarily utilizing 57Fe-Mössbauer spectroscopy, to study iron enzymes that play vital roles in various biological processes. Krebs's work extensively investigates enzymes that utilize non-heme iron cofactors to activate dioxygen for oxidation reactions, as well as enzymes containing iron-sulfur clusters, such as Radical-SAM enzymes. His research provides detailed insights into the reaction mechanisms of these enzymes, including the characterization of reaction intermediates and the understanding of their structural and functional diversity. His contributions have advanced the understanding of iron's role in enzymatic catalysis, with implications for biochemistry and potential applications in medicine and biotechnology.

Research topics

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

Selected publications

• Abstract 6799 The Winding Road to the Novel Mechanism of Microbial Ethylene-Forming Enzyme

  Journal of Biological Chemistry · 2026-05-01

  articleOpen access
  PublisherDOI

• Experimental Evidence for Radical-Polar Crossover in Competition with, Rather Than Preceding, Alkene Formation by Microbial Ethylene-Forming Enzyme

  Journal of the American Chemical Society · 2025-11-18

  articleOpen accessCorresponding

  Ethylene-forming enzyme (EFE) catalyzes a reaction that sets it apart from other iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases. In this reaction, all four oxidizing equivalents of O2 are unleashed upon 2OG, fragmenting it to ethylene (from C3 and C4) and three fully oxidized C1 equivalents (from C1, C2, and C5), while the would-be “prime substrate”, l-arginine, escapes unmodified. We previously proposed that ethylene formation proceeds by a radical-polar-crossover mechanism involving three unusual steps: (1) formal insertion of O2 between C1 and C2 of 2OG, forming a succinylperoxycarbonatoiron(II) complex and appending an additional oxygen to C1; (2) radical C–O coupling between a C3–C5-derived propionate-3-yl radical and a C1-derived Fe(III)-coordinated carbonate; and (3) polar fragmentation of the resultant (2-carboxyethyl)carbonatoiron(II) complex to ethylene, CO2, and carbonate. Here, we used isotopic labeling to distinguish the three C1 products and stopped-flow infrared (FTIR) spectroscopy to track their formation. The results confirm the prediction that C1 is not directly converted to CO2, implying that it must indeed become (bi)carbonate. Comparable kinetic data on the A198L variant, which produces ethylene and the abortive product, 3-hydroxypropionate, in similar quantities, reveal that these two products do not, as we had originally proposed, form in competing reactions of a common (2-carboxyethyl)carbonatoiron(II) intermediate. Rather, as suggested by a pair of computational studies separately led by Sayfutyarova and Christov, ethylene is formed in competition with radical coupling by an olefin-forming fragmentation that reduces the Fe(III) cofactor. In other words, crossover to the polar manifold thwarts rather than enables ethylene formation.

  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 access

  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

• 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

• 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

• 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

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

  Biochemistry · 2025-08-28

  articleOpen access

  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

• 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

• Steric Perturbation of the Grob-like Final Step of Ethylene-Forming Enzyme Enables 3-Hydroxypropionate and Propylene Production

  Journal of the American Chemical Society · 2024 · 16 citations

  • Chemistry
  • Stereochemistry
  • Medicinal chemistry

  )-methyl-2OG, presumably by allowing the otherwise sterically disfavored antiperiplanar conformation of the Grob intermediate bearing the extra methyl group. The results provide additional evidence for a polar-concerted ethylene-yielding step and thus for the proposed radical-polar crossover via substrate-radical coupling to the Fe(III)-coordinated carbonate.

  PublisherDOI

Recent grants

• NIH Grant R01GM103268

  NIH · $1.3M · 2017

• Structure and function of intermediates involved in metalloenzymatic N-oygenation and oxidation reactions

  NSF · $390k · 2011–2015

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

  NIH · $1.0M · 2016–2021

• NIH Grant R01GM055365

  NIH · $3.7M · 2013

• Non-heme Fe(IV)-oxo Intermediates: Structure and Reactivity

  NSF · $595k · 2007–2011

Frequent coauthors

• J. Martin Bollinger

  Pennsylvania State University

  211 shared

• Boi Hanh Huynh

  181 shared

• Wei Jiang

  76 shared

• Dale E. Edmondson

  Emory University

  54 shared

• Eric W. Barr

  University of Pennsylvania

  48 shared

• Laura M. K. Dassama

  Stanford Medicine

  47 shared

• Alice S. Pereira

  46 shared

• Stephen J. Lippard

  45 shared

Awards & honors

• Elected Fellow of the American Association for the Advanceme…
• SBIC Early Career Award (2012)
• Pfizer Award in Enzyme Chemistry (2008)
• Kavli Fellow of the National Academy of Sciences (2007)
• Camille Dreyfus Teacher Scholar (2006-2011)