William E. Broderick

Cysteine is a vital amino acid with wide biological utility, serving functions in catalysis, ligand binding, and maintenance of protein structures. In a subset of natural products, termed ribosomally synthesized and post-translationally modified peptides (RiPPs), multiple subclasses are defined by the enzymatic modification of cysteine residues to form thioether bridges. However, RiPPs are short peptides and experimental validation of enzymatically installed thioether crosslinks on large protein substrates is limited. We hypothesized that cysteine-rich proteins whose genes are adjacent to those for enzymes predicted to catalyze RiPP-like modifications might be substrates for such modifications. Thus, we set out to investigate cysteine-rich prokaryotic proteomes for such examples. A bioinformatic analysis identified methanogenic archaea as suitable candidates for further investigation due to the high overall proteome cysteine content, and through targeted proteome scanning, we identified a suitable biosynthetic gene cluster (BGC; Crp) from Methanococcus voltae to characterize. Purification and reconstitution of an annotated radical S-adenosyl-l-methionine (RS) enzyme, CrpC, confirmed that the protein binds three [4Fe-4S] clusters, two of which are in a SPASM domain. Enzyme assays determined that CrpC is active after reconstitution and modifies a cysteine-rich protein substrate in the same BGC, CrpA. Mass spectrometry and chemical derivatization of modified CrpA indicates that CrpC installs ranthionine crosslinks onto CrpA. This research provides a new path for the discovery of intriguing BGCs and post-translational modifications and provides a foundation for the complete structural elucidation and assessment of the native biological function of these modified cysteine-rich proteins.

Radical S -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.

The organometallic H-cluster of the [FeFe]-hydrogenase is assembled in vivo through a complex process requiring the action of three dedicated maturation enzymes, HydG, HydE, and HydF, as well as the aminomethyl-lipoyl-H-protein (H_met) of the glycine cleavage system (GCS). Here we probe the role of HydF and its [4Fe-4S] cluster in [FeFe]-hydrogenase maturation by using a defined semisynthetic approach in which [Fe^I₂(μ-SH)₂(CO)₄(CN)₂]^2- ([2Fe]_E) is used to bypass HydE and HydG, and GCS components are used in place of cell lysate. We show that inclusion of the iron-sulfur carrier protein NfuA and the high-CO-affinity myoglobin variant Mb^H64L provides dramatically improved hydrogenase activities up to 828 μmol/min/mg, equivalent to the best reported activities for Chlamydomonas reinhardtii [FeFe]-hydrogenase isolated from the native organism. Apo-HydF lacking a [4Fe-4S] cluster provides very little hydrogenase activity; however, full maturation is restored with the addition of NfuA, which we demonstrate reconstitutes the [4Fe-4S] cluster of HydF. In addition, a HydF variant lacking a [4Fe-4S] cluster by changing two cysteine ligands to alanine is completely unable to support either semisynthetic maturation using [2Fe]_E, or full maturation using HydG and HydE, even in the presence of NfuA, demonstrating that the HydF [4Fe-4S] cluster is absolutely essential for [FeFe]-hydrogenase maturation. The possibility that the HydF [4Fe-4S] cluster plays a role in direct binding of [2Fe]_E is negated by our results with the HydF^D311C variant, which demonstrate that the labile Asp311 cluster ligand is not essential for [2Fe]_E binding and HydA maturation. We therefore conclude that [2Fe]_E binds HydF adjacent to, but not directly coordinated to, the [4Fe-4S] cluster. The HydF [4Fe-4S] cluster is proposed to be essential due to its impact on the [2Fe]_E binding orientation and the ability of the HydF/[2Fe]_E complex to form productive interactions with H_met or the H_met/T-protein complex during DTMA ligand biosynthesis.

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.

Radical enzymes, including the evolutionarily ancient glycyl radical enzyme (GRE) family, catalyze chemically challenging reactions that are involved in a myriad of important biological processes. All GREs possess an essential, conserved backbone glycine that forms a stable catalytically essential α-carbon radical. Through close examination of the GRE family, we unexpectedly identified hundreds of noncanonical GRE homologues that encode either an alanine, serine, or threonine in place of the catalytic glycine residue. Contrary to a long-standing belief, we experimentally demonstrate that these aminoacyl radical enzymes (AAREs) form stable α-carbon radicals on the three cognate residues when activated by partner activating enzymes. The previously unrecognized AAREs are widespread in microbial genomes, highlighting their biological importance and potential for exhibiting new reactivity. Collectively, these studies expand the known radical chemistry of living systems while raising questions about the evolutionary emergence of the AAREs.

OspD is a radical S-adenosyl-l-methionine (SAM) peptide epimerase that converts an isoleucine (Ile) and valine (Val) of the OspA substrate to d-amino acids during biosynthesis of the ribosomally synthesized and post-translationally modified peptide (RiPP) natural product landornamide A. OspD is proposed to carry out this reaction via α-carbon (Cα) H-atom abstraction to form a peptidyl Cα radical that is stereospecifically quenched by hydrogen atom transfer (HAT) from a conserved cysteine (Cys). Here we use site-directed mutagenesis, freeze-quench trapping, isotopic labeling, and electron paramagnetic resonance (EPR) spectroscopy to provide new insights into the OspD catalytic mechanism including the direct observation of the substrate peptide Cα radical intermediate. The putative quenching Cys334 was changed to serine to generate an OspD C334S variant impaired in HAT quenching. The reaction of reduced OspD C334S with SAM and OspA freeze-quenched at 15 s exhibits a doublet EPR signal characteristic of a Cα radical coupled to a single β-H. Using isotopologues of OspA deuterated at either Ile or Val, or both Ile and Val, reveals that the initial Cα radical intermediate forms exclusively on the Ile of OspA. Time-dependent freeze quench coupled with EPR spectroscopy provided evidence for loss of the Ile Cα radical concomitant with gain of a Val Cα radical, directly demonstrating the N-to-C directionality of epimerization by OspD. These results provide direct evidence for the aforementioned OspD-catalyzed peptide epimerization mechanism via a central Cα radical intermediate during RiPP maturation of OspA, a mechanism that may extend to other proteusin peptide epimerases.

^1/2H and ¹³C hyperfine coupling constants to 5'-deoxyadenosyl (5'-dAdo•) radical trapped within the active site of the radical S-adenosyl-l-methionine (SAM) enzyme, pyruvate formate lyase-activating enzyme (PFL-AE), both in the absence of substrate and the presence of a reactive peptide-model of the PFL substrate, are completely characteristic of a classical organic free radical whose unpaired electron is localized in the 2pπ orbital of the sp² C5'-carbon (J. Am. Chem. Soc. 2019, 141, 12139-12146). However, prior electron-nuclear double resonance (ENDOR) measurements had indicated that this 5'-dAdo• free radical is never truly "free": tight van der Waals contact with its target partners and active-site residues guide it in carrying out the exquisitely precise, regioselective reactions that are hallmarks of RS enzymes. Here, our understanding of how the active site chaperones 5'-dAdo• is extended through the finding that this apparently unexceptional organic free radical has an anomalous g-tensor and exhibits significant ⁵⁷Fe, ¹³C, ¹⁵N, and ²H hyperfine couplings to the adjacent, isotopically labeled, methionine-bound [4Fe-4S]²⁺ cluster cogenerated with 5'-dAdo• during homolytic cleavage of cluster-bound SAM. The origin of the ⁵⁷Fe couplings through nonbonded radical-cluster contact is illuminated by a formal exchange-coupling model and broken symmetry-density functional theory computations. Incorporation of ENDOR-derived distances from C5'(dAdo•) to labeled-methionine as structural constraints yields a model for active-site positioning of 5'-dAdo• with a short, nonbonded C5'-Fe distance (∼3 Å). This distance involves substantial motion of 5'-dAdo• toward the unique Fe of the [4Fe-4S]²⁺ cluster upon S-C(5') bond-cleavage, plausibly an initial step toward formation of the Fe-C5' bond of the organometallic complex, Ω, the central intermediate in catalysis by radical-SAM enzymes.

Enzymes of the radical S -adenosyl- l -methionine (radical SAM, RS) superfamily, the largest in nature, catalyze remarkably diverse reactions initiated by H-atom abstraction. Glycyl radical enzyme activating enzymes (GRE-AEs) are a growing class of RS enzymes that generate the catalytically essential glycyl radical of GREs, which in turn catalyze essential reactions in anaerobic metabolism. Here, we probe the reaction of the GRE-AE pyruvate formate-lyase activating enzyme (PFL-AE) with the peptide substrate RVSG ₇₃₄ YAV, which mimics the site of glycyl radical formation on the native substrate, pyruvate formate-lyase. Time-resolved freeze-quench electron paramagnetic resonance spectroscopy shows that at short mixing times reduced PFL-AE + SAM reacts with RVSG ₇₃₄ YAV to form the central organometallic intermediate, Ω, in which the adenosyl 5′C is covalently bound to the unique iron of the [4Fe–4S] cluster. Freeze-trapping the reaction at longer times reveals the formation of the peptide G ₇₃₄ • glycyl radical product. Of central importance, freeze-quenching at intermediate times reveals that the conversion of Ω to peptide glycyl radical is not concerted. Instead, homolysis of the Ω Fe–C5′ bond generates the nominally “free” 5′-dAdo• radical, which is captured here by freeze-trapping. During cryoannealing at 77 K, the 5′-dAdo• directly abstracts an H-atom from the peptide to generate the G ₇₃₄ • peptide radical trapped in the PFL-AE active site. These observations reveal the 5′-dAdo• radical to be a well-defined intermediate, caught in the act of substrate H-atom abstraction, providing new insights into the mechanistic steps of radical initiation by RS enzymes.

The [FeFe]-hydrogenase can be matured using a defined semisynthetic approach in which the synthetic complex [2Fe]_Ebypasses the need for HydE and HydG, and glycine cleavage system components synthesize DTMA on [2Fe]_Ewhile it is bound to HydF.

The radical S-adenosyl methionine (SAM)enzymesuperfamily has widespread roles in hydrogen atom abstraction reactionsof crucial biological importance. In these enzymes, reductive cleavageof SAM bound to a [4Fe-4S](1+) cluster generates the 5 '-deoxyadenosylradical (5 '-dAdo center dot) which ultimately abstracts an H atomfrom the substrate. However, overwhelming experimental evidence hassurprisingly revealed an obligatory organometallic intermediate omega exhibiting an Fe-C5 '-adenosyl bond, whose properties are thetarget of this theoretical investigation. We report a readily applied,two-configuration version of broken symmetry DFT, denoted 2C-DFT,designed to allow the accurate description of the hyperfine couplingconstants and g-tensors of an alkyl group bound to a multimetalliciron-sulfur cluster. This approach has been validated by theexcellent agreement of its results both with those of multiconfigurationalcomplete active space self-consistent field computations for a seriesof model complexes and with the results from electron nuclear double-resonance/electronparamagnetic resonance spectroscopic studies for the crystallographicallycharacterized complex, M-CH3, a [4Fe-4S] clusterwith a Fe-CH3 bond. The likewise excellent agreementbetween spectroscopic results and 2C-DFT computations for omega confirm its identity as an organometallic complex with a bond betweenan Fe of the [4Fe-4S] cluster and C5 ' of the deoxyadenosylmoiety, as first proposed.