Richard J. Jodts

The MbnBC enzyme complex converts cysteine residues in a peptide substrate, MbnA, to oxazolone/thioamide groups during the biosynthesis of copper chelator methanobactin (Mbn). MbnBC belongs to the mixed-valent diiron oxygenase (MVDO) family, of which members use an Fe(II)Fe(III) cofactor to react with dioxygen for substrate modification. Several crystal structures of the inactive Fe(III)Fe(III) form of MbnBC alone and in complex with MbnA have been reported, but a mechanistic understanding requires determination of the oxidation states of the crystallographically observed Fe ions in the catalytically active Fe(II)Fe(III) state, along with the site of MbnA binding. Here, we have used electron nuclear double resonance (ENDOR) spectroscopy to determine such structural and electronic properties of the active site, in particular, the mode of substrate binding to the MV state, information not accessible by X-ray crystallography alone. The oxidation states of the two Fe ions were determined by ¹⁵N ENDOR analysis. The presence and locations of both bridging and terminal exogenous solvent ligands were determined using ¹H and ²H ENDOR. In addition, ²H ENDOR using an isotopically labeled MbnA substrate indicates that MbnA binds to the Fe(III) ion of the cluster via the sulfur atom of its N-terminal modifiable cysteine residue, with displacement of a coordinated solvent ligand as shown by complementary ¹H ENDOR. These results, which underscore the utility of ENDOR in studying MVDOs, provide a molecular picture of the initial steps in Mbn biosynthesis.

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.

Members of the radical S-adenosyl-L-methionine (SAM) enzyme superfamily initiate a broad spectrum of radical transformations through reductive cleavage of SAM by a [4Fe- 4S]1+ cluster it coordinates to generate the reactive 5 & PRIME;- deoxyadenosyl radical (5 & PRIME;-dAdo & BULL;). However, 5 & PRIME;-dAdo & BULL; is not directly liberated for reaction and instead binds to the unique Fe of the cluster to create the catalytically competent S = 1/2 organometallic intermediate omega. An alternative mode of reductive SAM cleavage, especially seen photochemically, instead liberates CH3 & BULL;, which forms the analogous S = 1/2 organometallic intermediate with an Fe-CH3 bond, omega M. The presence of a covalent Fe-C bond in both structures was established by the ENDOR observation of 13C and 1H hyperfine couplings to the alkyl groups that show isotropic components indicative of Fe-C bond covalency. The synthetic [Fe4S4]3+-CH3 cluster, M-CH3, is a crystallographically characterized analogue to omega M that exhibits the same [Fe4S4]3+ cluster state as omega and omega M, and thus an analysis of its spectroscopic properties?and comparison with those of omega and omega M?can be grounded in its crystal structure. We report cryogenic (2 K) EPR and 13C/1/2H ENDOR measurements on isotopically labeled M-CH3. At low temperatures, the complex exhibits EPR spectra from two distinct conformers/subpopulations. ENDOR shows that at 2 K, one contains a static methyl, but in the other, the methyl undergoes rapid tunneling/hopping rotation about the Fe-CH3 bond. This generates an averaged hyperfine coupling tensor whose analysis requires an extended treatment of rotational averaging. The methyl group 13C/1/2H hyperfine couplings are compared with the corresponding values for omega and omega M.

Significance: Methanobactins (Mbns), copper-binding peptidic compounds produced by some bacteria, are candidate therapeutics for human diseases of copper overload. The paired oxazolone-thioamide bidentate ligands of methanobactins are generated from cysteine residues in a precursor peptide, MbnA, by the MbnBC enzyme complex. MbnBC activity depends on the presence of iron and oxygen, but the catalytically active form has not been identified. Here, we provide evidence that a dinuclear Fe(II)Fe(III) center in MbnB, which is the only representative of a >13,000-member protein family to be characterized, is responsible for this reaction. These findings expand the known roles of diiron enzymes in biology and set the stage for mechanistic understanding, and ultimately engineering, of the MbnBC biosynthetic complex.

Radical S-adenosyl-l-methionine (SAM) enzymes employ a [4Fe–4S] cluster and SAM to initiate diverse radical reactions via either H-atom abstraction or substrate adenosylation. Here we use freeze-quench techniques together with electron paramagnetic resonance (EPR) spectroscopy to provide snapshots of the reaction pathway in an adenosylation reaction catalyzed by the radical SAM enzyme pyruvate formate-lyase activating enzyme on a peptide substrate containing a dehydroalanine residue in place of the target glycine. The reaction proceeds via the initial formation of the organometallic intermediate Ω, as evidenced by the characteristic EPR signal with g_∥ = 2.035 and g_⊥ = 2.004 observed when the reaction is freeze-quenched at 500 ms. Thermal annealing of frozen Ω converts it into a second paramagnetic species centered at g_iso = 2.004; this second species was generated directly using freeze-quench at intermediate times (∼8 s) and unequivocally identified via isotopic labeling and EPR spectroscopy as the tertiary peptide radical resulting from adenosylation of the peptide substrate. An additional paramagnetic species observed in samples quenched at intermediate times was revealed through thermal annealing while frozen and spectral subtraction as the SAM-derived 5′-deoxyadenosyl radical (5′-dAdo•). The time course of the 5′-dAdo• and tertiary peptide radical EPR signals reveals that the former generates the latter. These results thus support a mechanism in which Ω liberates 5′-dAdo• by Fe–C5′ bond homolysis, and the 5′-dAdo• attacks the dehydroalanine residue of the peptide substrate to form the adenosylated peptide radical species. The results thus provide a picture of a catalytically competent 5′-dAdo• intermediate trapped just prior to reaction with the substrate.

In this report, we investigate the physical and chemical properties of monocopper Cu(I) superoxo and Cu(II) peroxo and hydroperoxo complexes. These are prepared by cryoreduction/annealing of the parent [LCu^I(O₂)]⁺ Cu(I) dioxygen adducts with the tripodal, N₄-coordinating, tetradentate ligands L = ^PVtmpa, ^DMMtmpa, TMG₃tren and are best described as [LCu^II(O₂^•–)]⁺ Cu(II) complexes that possess end-on (η¹-O₂^•–) superoxo coordination. Cryogenic γ-irradiation (77 K) of the EPR-silent parent complexes generates mobile electrons from the solvent that reduce the [LCu^II(O₂^•–)]⁺ within the frozen matrix, trapping the reduced form fixed in the structure of the parent complex. Cryoannealing, namely progressively raising the temperature of a frozen sample in stages and then cooling back to low temperature at each stage for examination, tracks the reduced product as it relaxes its structure and undergoes chemical transformations. We employ EPR and ENDOR (electron–nuclear double resonance) as powerful spectroscopic tools for examining the properties of the states that form. Surprisingly, the primary products of reduction of the Cu(II) superoxo species are metastable cuprous superoxo [LCu^I(O₂^•–)]⁺ complexes. During annealing to higher temperatures this state first undergoes internal electron transfer (IET) to form the end-on Cu(II) peroxo state, which is then protonated to form Cu(II)–OOH species. This is the first time these methods, which have been used to determine key details of metalloenzyme catalytic cycles and are a powerful tools for tracking PCET reactions, have been applied to copper coordination compounds.

In nature, methane is oxidized to methanol by two enzymes, the iron-dependent soluble methane monooxygenase (sMMO) and the copper-dependent particulate MMO (pMMO). While sMMO’s diiron metal active site is spectroscopically and structurally well-characterized, pMMO’s copper sites are not. Recent EPR and ENDOR studies have established the presence of two monocopper sites, but the coordination environment of only one has been determined, that within the PmoB subunit and denoted Cu_B. Moreover, this recent work only focused on a type I methanotrophic pMMO, while previous observations of the type II enzyme were interpreted in terms of the presence of a dicopper site. First, this report shows that the type II Methylocystis species strain Rockwell pMMO, like the type I pMMOs, contains two monocopper sites and that its Cu_B site has a coordination environment identical to that of type I enzymes. As such, for the full range of pMMOs this report completes the refutation of prior and ongoing suggestions of multicopper sites. Second, and of primary importance, EPR/ENDOR measurements (a) for the first time establish the coordination environment of the spectroscopically observed site, provisionally denoted Cu_C, in both types of pMMO, thereby (b) establishing the assignment of this site observed by EPR to the crystallographically observed metal-binding site in the PmoC subunit. Finally, these results further indicate that Cu_C is the likely site of biological methane oxidation by pMMO, a conclusion that will serve as a foundation for proposals regarding the mechanism of this reaction.

Some methane-oxidizing bacteria use the ribosomally synthesized, posttranslationally modified natural product methanobactin (Mbn) to acquire copper for their primary metabolic enzyme, particulate methane monooxygenase. The operons encoding the machinery to biosynthesize and transport Mbns typically include genes for two proteins, MbnH and MbnP, which are also found as a pair in other genomic contexts related to copper homeostasis. While the MbnH protein, a member of the bacterial diheme cytochrome c peroxidase (bCcP)/MauG superfamily, has been characterized, the structure and function of MbnP, the relationship between the two proteins, and their role in copper homeostasis remain unclear. Biochemical characterization of MbnP from the methanotroph Methylosinus trichosporium OB3b now reveals that MbnP binds a single copper ion, present in the +1 oxidation state, with high affinity. Copper binding to MbnP in vivo is dependent on oxidation of the first tryptophan in a conserved WxW motif to a kynurenine, a transformation that occurs through an interaction of MbnH with MbnP. The 2.04-Å-resolution crystal structure of MbnP reveals a unique fold and an unusual copper-binding site involving a histidine, a methionine, a solvent ligand, and the kynurenine. Although the kynurenine residue may not serve as a Cu^I primary-sphere ligand, being positioned ∼2.9 Å away from the Cu^I ion, its presence is required for copper binding. Genomic neighborhood analysis indicates that MbnP proteins, and by extension kynurenine-containing copper sites, are widespread and may play diverse roles in microbial copper homeostasis. Significance Copper is particularly important in the metabolism of methanotrophs, methane-consuming bacteria that have potential applications in converting methane, a greenhouse gas, to useful fuels and chemicals. How methanotrophs and other bacteria acquire and distribute copper, which is toxic in excess, is not well understood. Here we identify and characterize a previously unknown copper-binding protein implicated in methanotroph copper homeostasis. This protein, MbnP, binds copper with an unusual coordination sphere that includes kynurenine, a tryptophan oxidation product that is produced in vivo by a partner protein, MbnH. MbnP is the founding member of a widespread protein family with potentially broad roles in microbial copper homeostasis.

Catalysis by canonical radical S-adenosyl-l-methionine (SAM) enzymes involves electron transfer (ET) from [4Fe–4S]⁺ to SAM, generating an R₃S⁰ radical that undergoes regioselective homolytic reductive cleavage of the S–C5′ bond to generate the 5′-dAdo· radical. However, cryogenic photoinduced S–C bond cleavage has regioselectively yielded either 5′-dAdo· or ·CH₃, and indeed, each of the three SAM S–C bonds can be regioselectively cleaved in an RS enzyme. This diversity highlights a longstanding central question: what controls regioselective homolytic S–C bond cleavage upon SAM reduction? We here provide an unexpected answer, founded on our observation that photoinduced S–C bond cleavage in multiple canonical RS enzymes reveals two enzyme classes: in one, photolysis forms 5′-dAdo·, and in another it forms ·CH₃. The identity of the cleaved S–C bond correlates with SAM ribose conformation but not with positioning and orientation of the sulfonium center relative to the [4Fe–4S] cluster. We have recognized the reduced-SAM R₃S⁰ radical is a (²E) state with its antibonding unpaired electron in an orbital doublet, which renders R₃S⁰ Jahn–Teller (JT)-active and therefore subject to vibronically induced distortion. Active-site forces induce a JT distortion that localizes the odd electron in a single priority S–C antibond, which undergoes regioselective cleavage. In photolytic cleavage those forces act through control of the ribose conformation and are transmitted to the sulfur via the S–C5′ bond, but during catalysis thermally induced conformational changes that enable ET from a cluster iron generate dominant additional forces that specifically select S–C5′ for cleavage. This motion also can explain how 5′-dAdo· subsequently forms the organometallic intermediate Ω.

Spore photoproduct lyase is a radical S-adenosyl-l-methionine (SAM) enzyme with the unusual property that addition of SAM to the [4Fe-4S]¹⁺ enzyme absent substrate results in rapid electron transfer to SAM with accompanying homolytic S–C5′ bond cleavage. Herein, we demonstrate that this unusual reaction forms the organometallic intermediate Ω in which the unique Fe atom of the [4Fe-4S] cluster is bound to C5′ of the 5′-deoxyadenosyl radical (5′-dAdo^•). During catalysis, homolytic cleavage of the Fe–C5′ bond liberates 5′-dAdo^• for reaction with substrate, but here, we use Ω formation without substrate to determine the thermal stability of Ω. The reaction of Geobacillus thermodenitrificans SPL (GtSPL) with SAM forms Ω within ∼15 ms after mixing. By monitoring the decay of Ω through rapid freeze–quench trapping at progressively longer times we find an ambient temperature decay time of the Ω Fe–C5′ bond of τ ≈ 5–6 s, likely shortened by enzymatic activation as is the case with the Co–C5′ bond of B₁₂. We have further used hand quenching at times up to 10 min, and thus with multiple SAM turnovers, to probe the fate of the 5′-dAdo^• radical liberated by Ω. In the absence of substrate, Ω undergoes low-probability conversion to a stable protein radical. The WT enzyme with valine at residue 172 accumulates a Val^•; mutation of Val172 to isoleucine or cysteine results in accumulation of an Ile^• or Cys^• radical, respectively. The structures of the radical in WT, V172I, and V172C variants have been established by detailed EPR/DFT analyses.