About

Brian M. Hoffman is a professor in the Interdisciplinary Biological Sciences Graduate Program at Northwestern University. He holds a PhD from the California Institute of Technology. His research focuses on determining the catalytic mechanisms of metalloenzymes through the development and implementation of electron-nuclear double resonance (ENDOR) spectroscopy, a technique that combines NMR and EPR to analyze active-site nuclei by monitoring the metal centers. This method allows for the characterization of enzyme active site composition, electronic, and geometric structures, particularly for catalytic intermediates trapped during enzyme reactions. His ongoing projects include studies of biological nitrogen fixation by nitrogenase, radical SAM enzyme catalysis, methane oxidation by Cu methane monooxygenase, and in vivo speciation of Mn 2+ ions. His work also involves parallel studies of synthetic biomimetic complexes to identify intermediates during catalysis and explore their dynamic properties. Hoffman’s research aims to reveal enzyme mechanisms and contribute to understanding key biological processes such as nitrogen fixation, radical reactions, methane mitigation, and metal ion roles in biological systems.

Research topics

• Chemistry
• Organic chemistry
• Stereochemistry
• Crystallography
• Ecology
• Nanotechnology
• Photochemistry
• Biochemistry
• Inorganic chemistry
• Agronomy
• Environmental chemistry
• Atomic physics
• Physics
• Biology
• Medicinal chemistry
• Combinatorial chemistry
• Nuclear magnetic resonance
• Biochemical engineering
• Computational chemistry

Selected publications

• The radical SAM enzyme EpeE exhibits distinct site reactivity during the biosynthesis of the RiPP natural product epipeptide

  Proceedings of the National Academy of Sciences · 2026-03-18

  articleOpen access

  -adenosyl-l-methionine (SAM) enzymes figure prominently in the formation of ribosomally synthesized and posttranslationally modified peptides (RiPPs), where they catalyze peptide modifications including epimerization, thioether crosslink formation, and peptide backbone splicing. Here, we use rapid freeze-quench trapping together with electron paramagnetic resonance and electron-nuclear double resonance techniques to probe the mechanistic steps of the two epimerization reactions catalyzed by the radical SAM enzyme EpeE during conversion of its peptide substrate to the epipeptide natural product. Use of the EpeE C223S variant facilitated trapping and characterization of Cα radical intermediates, supporting a central role for C223 in the proposed epimerization mechanism. We showed that both wild-type and C223S EpeE with bound SAM and peptide substrate form the organometallic intermediate Ω upon reaction, and that thermal annealing of Ω results in conversion to an organic radical intermediate. Freeze-quenching at longer times allowed us to directly trap the organic radical intermediate, and isotopic labeling together with use of substrate variants allowed for detailed characterization of the substrate radical intermediates. The results revealed that while LC-MS enzymatic assays point to Ile12 as the initial site of epimerization, freeze-quench EPR reveals that Val4 is the preferred site for initial Cα radical formation. These apparently conflicting results were resolved by the observation that the Ile12 Cα radical is more efficiently quenched to form the d-Ile, thus providing insights into the determinants for substrate binding and epimerization by EpeE.

  PublisherOA PDFDOI

• Sulfite Is Not Required for N ₂ Reduction Catalyzed by Mo-Nitrogenase

  Journal of the American Chemical Society · 2026-04-17

  article

  Mo-nitrogenase catalyzes the reduction of dinitrogen (N2) to two ammonia (NH3) at the active-site FeMo-cofactor. Substrate activation requires the accumulation of three or four electrons and protons as two Fe-bound hydrides and is coupled to obligatory H2 release through reductive hydride elimination. Subsequent delivery of four or five additional electrons and protons to the bound N2 yields two NH3 molecules. Increasing evidence suggests that at least one belt sulfide within FeMo-cofactor is dynamically involved in the catalytic cycle. A recent report further proposed that sulfite (SO32–) is required for N2 reduction, with sulfite binding required for NH3 release and a subsequent six-electron reduction of the bound sulfite to regenerate the resting cofactor. To test this proposal, we conducted turnover studies of Mo-nitrogenase under sulfite-free conditions using a reduced viologen as reductant and protein preparations devoid of dithionite or sulfite. Under these conditions, nitrogenase effectively catalyzed both N2 reduction and proton reduction, exhibiting steady-state turnover under N2 for 6 min, with a turnover number exceeding 150, approaching that observed with dithionite as reductant. The same H2-formed/N2-reduced ratio was observed whether dithionite or the viologen species was used as reductant. Further, EPR spectroscopic analyses showed that the FeMo-cofactor returned to its resting state after multiple catalytic cycles in the absence of sulfite. Finally, physiological bypass of sulfite formation does not affect the capacity for diazotrophic growth of the model nitrogen-fixing organism Azotobacter vinelandii. These results demonstrate that sulfite is not required for Mo-nitrogenase-catalyzed N2 reduction either in vitro or in vivo.

  PublisherDOI

• Nitric Oxide restricts iron availability and induces quorum sensing in Streptococcus pyogenes

  Redox Biology · 2025-06-04 · 4 citations

  articleOpen access

  Nitric oxide (NO) is a free radical signaling molecule with multiple biological functions. As part of the innate immune system, NO has antimicrobial properties playing an important role in host defense. Mechanisms of NO cytotoxicity result from its ability to bind metals and inhibit enzyme function or by increasing nitrosative and oxidative stress within cells. One of the primary biological targets of NO is the chelatable iron pool (CIP) which is quantitatively converted to dinitrosyliron complexes (DNIC) when it reacts with NO. Despite the numerous purported mechanisms attributed to NO's bactericidal properties, DNIC formation and its ability to restrict iron bioavailability from pathogenic bacteria has not been directly tested. Streptococcus pyogenes is a human pathogen that causes a range of diseases spanning from pharyngitis and impetigo to soft tissue necrosis and toxic shock. S. pyogenes employs the Rgg2/Rgg3 quorum sensing (QS) system to regulate aspects of its virulence potential, including biofilm formation, lysozyme resistance, and modulation of host innate immune response. Previous studies found that iron and manganese restriction induced Rgg2/Rgg3 QS, leading us to test whether NO-dependent iron restriction mediated by DNIC formation was sufficient to induce QS and related iron-starvation phenotypes. Here, we demonstrate that DNIC are formed in S. pyogenes exposed to physiologically relevant NO concentrations. The DNIC are formed from the CIP, and formation led to a significant reduction in the CIP, which correlated to a concomitant activation of QS and iron-regulated gene expression. These studies are the first to demonstrate that restriction of iron bioavailability mediated by DNIC formation is a functional mechanism by which NO can regulate QS, gene expression, and cell growth in bacteria.

  PublisherDOI

• Simultaneous occupancy of Cu _C and Cu _D in the ammonia monooxygenase active site

  Chemical Science · 2025-12-18 · 1 citations

  articleOpen access

  sites, separating them by ∼8.0 Å. The results underscore the importance of studying these enzymes in their native environments across species to resolve conserved and divergent molecular features.

  PublisherOA PDFDOI

• Toward a Unified Kinetic Model of Nitrogenase Catalysis

  ACS Catalysis · 2025-10-15 · 5 citations

  article

  The microbial enzyme nitrogenase catalyzes the MgATP-dependent reduction of N2 to 2NH3, a transformation central to the global nitrogen cycle. While the canonical Thorneley–Lowe (TL) kinetic model has long served as a mechanistic framework, it does not incorporate several recent insights. Here, we present an updated kinetic model for Mo-nitrogenase that incorporates these new findings. A significant insight is that electron transfer (ET) from the reduced Fe protein to the FeMo-cofactor is gated by MgATP-dependent conformational transitions and can be described as a probabilistic event that is dependent on the ligand bound to the active-site metallocofactor. The updated kinetic model quantitatively reproduces steady-state product formation rates across a broad range of experimental conditions, yielding revised estimates for key rate constants. It is demonstrated that under N2 turnover, the probability of productive ET to the active site decreases by ∼60%, resulting in a significant fraction of Fe protein cycles that are unproductive for electron delivery. This mechanistic feature explains the observed rate limitation in N2 reduction and implies a revised minimum energetic cost of approximately 25 MgATP per N2 reduced. Integrating these new features into the revised kinetic model provides a more complete and usable foundation for understanding nitrogenase catalysis.

  PublisherDOI

• Spectroscopic Interrogation of Thiolate Hydrogen Bonding in the CO Sensor Protein CooA: Implications for Thiolate Ligation Stability and Cytochrome P450 Function

  Chemistry - A European Journal · 2025-05-21 · 1 citations

  articleOpen access

  Abstract Cysteine (Cys) thiolate coordination in hemoproteins is a unique ligation motif found in enzymes and small molecule sensors. It is posited that divergence between these two functional classes of heme thiolate proteins is a result of distinct hydrogen bonding (H‐bonding) interactions with the axial Cys(thiolate) ligand. To further test this hypothesis, we report a spectroscopic analysis of thiolate H‐bonding in CooA, a carbon monoxide‐sensing heme protein from Rhodospirillum rubrum that is known to switch between Cys 75 (thiolate) and histidine coordination upon reduction. We generated CooA variants with alterations at two residues, Asn 42 and His 77 , which are postulated to influence H‐bonding to Cys 75 on the basis of an Fe(II) CooA structure. Using a combination of electronic absorption, electron paramagnetic resonance (EPR), and electron nuclear double resonance (ENDOR) spectroscopies, we identified several CooA variants that exhibit changes in thiolate donor strength and propose an H‐bonding model in which Asn 42 orients His 77 for optimal H‐bonding with Cys 75 in Fe(III) CooA. Further, we spectroscopically characterize pyrrolidine‐bound CYP119, a cytochrome P450, to mimic the first coordination sphere of Fe(III) CooA. Our data unequivocally show that CooA contains a stronger thiolate‐Fe bond than the pyrrolidine‐bound CYP119, suggesting that Cys(thiolate) H‐bonding interactions in CooA are significantly weaker. These results support the hypothesis that thiolate H‐bonding is a significant differentiator between the two classes of heme thiolate proteins.

  PublisherOA PDFDOI

• Abstract 5182: Transcriptomic analysis of mouse models of lung adenocarcinoma with different metastatic phenotypes

  Cancer Research · 2025-04-21

  article

  Introduction: Lung cancer is the leading cause of cancer deaths worldwide with most deaths attributed to metastatic disease. Existing mouse models of lung adenocarcinoma (LUAD) rarely become metastatic, limiting their usefulness for basic and pre-clinical research aimed at identifying mechanisms of metastatic disease. We have developed two novel mouse models of LUAD, KPDT-1 and KPDT-2, that feature cell type-specific inducible oncogenic KrasG12D, loss of Trp53 function, and deleted/truncated Dicer1. The survival times, rate of tumor progression, and metastatic phenotypes of these models depend on the cell types that harbor the genetic alterations. We performed transcriptomics (mRNA and microRNA) to investigate the molecular and cellular drivers underlying the observed differences in the models. Methods and Results: KPDT-1 and KPDT-2 models were generated by adding mutations in Dicer1, an RNAse III enzyme within the microRNA (miRNA) biosynthesis pathway, to a mouse model of Kras-driven LUAD. For KPDT-1, tumorigenesis was induced by conditional expression of an oncogenic KrasG12D allele, deletion of both alleles of Trp53 and deletion of one allele of Dicer1 in club cells and the expression of a truncated Dicer1 allele in alveolar type II (ATII) cells. In KPDT-2 animals, the cell types were reversed. We observed decreased survival, accelerated tumor progression, and enhanced lymph node metastasis only in KPDT-1 animals. Induction of tumorigenesis in ATII cells and truncation of Dicer1 in club cells (KPDT-2) did not accelerate tumor progression. Differential expression analysis using DESeq2 revealed 206 differentially expressed genes and 45 differentially expressed microRNAs between the two models. Term enrichment analyses showed that genes upregulated in KPDT-1 animals are associated with microtubule movement and organization. The upregulated genes of KPDT-2 animals are enriched in functions related to metabolism. Six microRNAs were upregulated in the KPDT-1 model. These miRNAs were reported previously as being differentially expressed in other cancers, and 4 of them (miR-92a-3p, miR-542-5p, miR-449a-5p, and miR-296-5p) have been linked previously to metastasis. Conclusions: Through cell type-specific truncation/deletion of Dicer1, we have generated a new mouse model that rapidly develops Kras/Trp53-driven LUAD and metastatic disease. Our findings suggest that tumorigenesis and metastasis are influenced by miRNA-mediated communication between different cell types. Further investigation into the pathways involved in these models will give more insight into miRNA-mediated tumorigenesis and the underlying mechanisms of metastasis. We will continue to develop these models to support investigation of molecular mechanisms underlying metastasis. Citation Format: Naomi Mitchell-Hutchinson, Paige Ramkissoon, Julie Wells, Rick Maser, Tim Stodola, Brian Hoffman, Anne Marchini, Elaine Bechtel, Rosalinda Doty, Carol Bult. Transcriptomic analysis of mouse models of lung adenocarcinoma with different metastatic phenotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5182.

  PublisherDOI

• EPR spectroscopy reveals antioxidant manganese defenses in the Lyme disease pathogen <i>Borrelia burgdorferi</i>

  mBio · 2025-11-13 · 2 citations

  articleOpen access

  ABSTRACT Oxidative stress defense in aerobic bacteria relies on Mn-superoxide dismutase (MnSOD) and antioxidant Mn-metabolite complexes (H-Mn) to quench superoxide radicals (O 2 •− ). We investigated these antioxidant systems in Borrelia burgdorferi , the Mn-accumulating, Fe-independent Lyme disease pathogen. Using electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) spectroscopies, we tracked Mn² + partitioning between enzyme-bound (L-Mn) and metabolite-bound (H-Mn) pools in spirochetes at exponential and stationary phases. Results show that MnSOD neutralizes extracellular O 2 •− generated by γ-irradiation (a model for host immune attack); H-Mn neutralizes cytoplasmic O 2 •− and is a reservoir of labile Mn² + for metalating Mn-dependent enzymes. MnCl 2 supplementation in log phase B. burgdorferi restored radioresistance in ΔMnSOD mutants via H-Mn hyperaccumulation but induced toxicity in older, stationary phase cells as metabolites became depleted. These findings support an expanded oxidative-stress model in which H-Mn complements MnSOD and positions Mn homeostasis as a therapeutic target. Our approach highlights the utility of EPR and ENDOR in studying Mn-dependent pathogens. IMPORTANCE We employed electron paramagnetic resonance and electron-nuclear double resonance spectroscopies of Mn² + in intact Borrelia burgdorferi supplemented with MnCl 2 to track changes in the amounts of enzyme-bound Mn and substitutionally labile, antioxidant Mn-metabolite complexes. We measured the spirochete’s survivability to acute γ-irradiation, which simulates the respiratory burst of O 2 •− deployed as a critical weapon in the host’s innate immune response. While Mn-superoxide dismutase (MnSOD) has classically been viewed as the main defense against oxidative damage in B. burgdorferi , our study demonstrates that antioxidant Mn 2+ complexes with the metabolite components of H-Mn play a crucial antioxidant role, particularly when MnSOD is deficient. However, B. burgdorferi’s inability to safely store excess Mn in metabolite-depleted cells highlights novel metabolic vulnerabilities that could be exploited for managing Lyme disease.

  PublisherDOI

• In vitro maturation of fully active [FeFe]-hydrogenase in a defined system including the iron carrier NfuA

  Proceedings of the National Academy of Sciences · 2025-09-22 · 1 citations

  articleOpen access

  The [FeFe]-hydrogenase employs an active-site 6Fe H-cluster to catalyze the reversible reduction of protons to H 2 . A [4Fe-4S] subcluster of the H-cluster is synthesized by housekeeping iron-sulfur cluster assembly machinery, and then dedicated hydrogenase maturation enzymes, together with components of the glycine cleavage system, build and deliver a [2Fe] subcluster to generate the full H-cluster. Here, we report that the Escherichia coli iron-sulfur carrier protein NfuA supports in vitro maturation of fully active [FeFe]-hydrogenase, with H 2 production rates comparable to that of the in vivo - matured Chlamydomonas reinhardtii [FeFe]-hydrogenase ( Cr HydA). Inclusion of NfuA in the in vitro maturation process improves its efficacy by delivering the iron essential for formation of the [Fe II (cys)(CN)(CO) 2 ] – synthon at the dangler iron site of the HydG auxiliary cluster. NfuA serves an additional role in reconstituting and maintaining the catalytically essential iron-sulfur clusters on the maturase enzymes HydE, HydF, and HydG. Further inclusion of a high CO affinity myoglobin variant (Mb H64L ) sequesters free CO generated during the maturation process, minimizing formation of the CO-inhibited H ox -CO enzyme state, significantly increasing hydrogenase activity. The addition of NfuA and Mb H64L to the fully defined maturation system thus results in an in vitro [FeFe]-hydrogenase maturation system that generates highly active enzyme while providing insights into factors important to in vivo maturation.

  PublisherDOI

• Prophylactically Feeding Manganese to Drosophila Confers Sex-Specific Protection from Acute Ionizing Radiation Independent of MnSOD2 Levels

  Antioxidants · 2025-01-23 · 4 citations

  articleOpen access

  Ionizing radiation is a health threat to many, including warfighters, radiological emergency responders, radiotherapy patients, and astronauts. Despite this, no FDA-approved prophylactic medical countermeasures exist to attenuate the symptoms that occur from radiation exposure. Manganese has recently been shown to be critical for radioresistance in a wide range of organisms. In this study, we designed a stringent feeding method to test the prophylactic effects of dietary manganese on Drosophila’s lifespan before exposure to acute irradiation. We found that male flies have substantially lower radioresistance than females, but feeding with low doses of MnCl2 before acute irradiation exposure extends male survival to that of females. Whole animal electron paramagnetic resonance analyses showed males have lower amounts of high-symmetry manganese-metabolite antioxidant complexes (H-Mn) than females, but manganese supplementation increases H-Mn to female levels. Levels of mitochondrial free-radical scavenger manganese-superoxide-dismutase 2 (MnSOD2) did not increase after acute irradiation, nor did loss of MnSOD2 sensitize larvae to acute irradiation exposure. These data support that prophylactic manganese feeding is sufficient to increase survivorship in males subjected to acute irradiation, independent of MnSOD2 levels, indicating a role of antioxidant manganese-metabolite H-Mn complexes for radioprotection. Furthermore, this Drosophila feeding method could be used to identify additional radiation countermeasures.

  PublisherOA PDFDOI

Recent grants

• NIH Grant R01HL063203

  NIH · $6.1M · 2017

• NIH Grant R37HL013531

  NIH · $3.3M · 1998

• Star Porphyrazines

  NSF · $450k · 2005–2009

• Biophysical Studies of Metalloenzymes

  NSF · $597k · 2003–2008

• Biophysical Studies of Metalloenzymes

  NSF · $705k · 2024–2028

Frequent coauthors

• Dennis R. Dean

  Virginia Tech

  271 shared

• Roman Davydov

  Northwestern University

  262 shared

• Lance C. Seefeldt

  250 shared

• Joshua Telser

  Roosevelt University

  210 shared

• Anthony G. M. Barrett

  Imperial College London

  208 shared

• Hong-In Lee

  Kyungpook National University

  150 shared

• Mikhail Laryukhin

  National Eye Institute

  133 shared

• Peter E. Doan

  Northwestern University

  114 shared