Browsing by Author "Shepard, Eric M."

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Decarboxylation involving a ferryl, propionate, and a tyrosyl group in a radical relay yields heme b
(2018-03) Streit, Bennett R.; Celis Luna, Arianna I.; Moraski, Garrett C.; Shisler, Krista A.; Shepard, Eric M.; Rodgers, Kenton R.; Lukat-Rodgers, Gudrun S.; DuBois, Jennifer L.
The H2O2-dependent oxidative decarboxylation of coproheme III is the final step in the biosynthesis of heme b in many microbes. However, the coproheme decarboxylase reaction mechanism is unclear. The structure of the decarboxylase in complex with coproheme III suggested that the substrate iron, reactive propionates, and an active-site tyrosine convey a net 2e-/2H+ from each propionate to an activated form of H2O2 Time-resolved EPR spectroscopy revealed that Tyr-145 forms a radical species within 30 sec of the reaction of the enzyme-coproheme complex with H2O2 This radical disappeared over the next 270 sec, consistent with a catalytic intermediate. Use of the harderoheme III intermediate as substrate or substitutions of redox-active side chains (W198F, W157F, or Y113S) did not strongly affect the appearance or intensity of the radical spectrum measured 30 sec after initiating the reaction with H2O2, nor did it change the ~270 sec required for the radical signal to recede to â‰¤ 10% of its initial intensity. These results suggested Tyr-145 as the site of a catalytic radical involved in decarboxylating both propionates. Tyr-145 was accompanied by partial loss of the initially present Fe(III) EPR signal intensity, consistent with the possible formation of Fe(IV)=O. Site-specifically deuterated coproheme gave rise to a kinetic isotope effect of ~2 on the decarboxylation rate constant, indicating that cleavage of the propionate Cbeta-H bond was partly rate limiting. The inferred mechanism requires two consecutive hydrogen atom transfers, first from Tyr-145 to the substrate Fe/H2O2 intermediate and then from the propionate Cbeta-H to Tyr-145.
[FeFe]- and [NiFe]-hydrogenase diversity, mechanism, and maturation
(2014-11) Peters, John W.; Schut, Gerrit J.; Boyd, Eric S.; Mulder, David W.; Shepard, Eric M.; Broderick, Joan B.; King, Paul W.; Adams, Michael W. W.
The [FeFe]- and [NiFe]-hydrogenases catalyze the formal interconversion between hydrogen and protons and electrons, possess characteristic non-protein ligands at their catalytic sites and thus share common mechanistic features. Despite the similarities between these two types of hydrogenases, they clearly have distinct evolutionary origins and likely emerged from different selective pressures. [FeFe]-hydrogenases are widely distributed in fermentative anaerobic microorganisms and likely evolved under selective pressure to couple hydrogen production to the recycling of electron carriers that accumulate during anaerobic metabolism. In contrast, many [NiFe]-hydrogenases catalyze hydrogen oxidation as part of energy metabolism and were likely key enzymes in early life and arguably represent the predecessors of modern respiratory metabolism. Although the reversible combination of protons and electrons to generate hydrogen gas is the simplest of chemical reactions, the [FeFe]- and [NiFe]-hydrogenases have distinct mechanisms and differ in the fundamental chemistry associated with proton transfer and control of electron flow that also help to define catalytic bias. A unifying feature of these enzymes is that hydrogen activation itself has been restricted to one solution involving diatomic ligands (carbon monoxide and cyanide) bound to an Fe ion. On the other hand, and quite remarkably, the biosynthetic mechanisms to produce these ligands are exclusive to each type of enzyme. Furthermore, these mechanisms represent two independent solutions to the formation of complex bioinorganic active sites for catalyzing the simplest of chemical reactions, reversible hydrogen oxidation. As such, the [FeFe]- and [NiFe]-hydrogenases are arguably the most profound case of convergent evolution. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.
Identification and characterization of a novel member of the radical AdoMet enzyme superfamily and implications for the biosynthesis of the Hmd hydrogenase active site cofactor
(2009-11) McGlynn, Shawn E.; Boyd, Eric S.; Shepard, Eric M.; Lange, Rachel K.; Gerlach, Robin; Broderick, Joan B.; Peters, John W.
The genetic context, phylogeny, and biochemistry of a gene flanking the H2-forming methylene-H4-methanopterin dehydrogenase gene (hmdA), here designated hmdB, indicate that it is a new member of the radical S-adenosylmethionine enzyme superfamily. In contrast to the characteristic CX3CX2C or CX2CX4C motif defining this family, HmdB contains a unique CX5CX2C motif.
Impact of mineral and non-mineral sources of iron and sulfur on the metalloproteome of Methanosarcina barkeri
(American Society for Microbiology, 2024-07) Larson, James; Tokmina-Lukaszewska, Monika; Payne, Devon; Spietz, Rachel L.; Fausset, Hunter; Alam, Md Gahangir; Brekke, Brooklyn K.; Pauley, Jordan; Hasenoehrl, Ethan J.; Shepard, Eric M.; Boyd, Eric S.; Bothner, Brian
Methanogens often inhabit sulfidic environments that favor the precipitation of transition metals such as iron (Fe) as metal sulfides, including mackinawite (FeS) and pyrite (FeS2). These metal sulfides have historically been considered biologically unavailable. Nonetheless, methanogens are commonly cultivated with sulfide (HS-) as a sulfur source, a condition that would be expected to favor metal precipitation and thus limit metal availability. Recent studies have shown that methanogens can access Fe and sulfur (S) from FeS and FeS2 to sustain growth. As such, medium supplied with FeS2 should lead to higher availability of transition metals when compared to medium supplied with HS-. Here, we examined how transition metal availability under sulfidic (i.e., cells provided with HS- as sole S source) versus non-sulfidic (cells provided with FeS2 as sole S source) conditions impact the metalloproteome of Methanosarcina barkeri Fusaro. To achieve this, we employed size exclusion chromatography coupled with inductively coupled plasma mass spectrometry and shotgun proteomics. Significant changes were observed in the composition and abundance of iron, cobalt, nickel, zinc, and molybdenum proteins. Among the differences were alterations in the stoichiometry and abundance of multisubunit protein complexes involved in methanogenesis and electron transport chains. Our data suggest that M. barkeri utilizes the minimal iron-sulfur cluster complex and canonical cysteine biosynthesis proteins when grown on FeS2 but uses the canonical Suf pathway in conjunction with the tRNA-Sep cysteine pathway for iron-sulfur cluster and cysteine biosynthesis under sulfidic growth conditions.
Proteomic Analysis of Methanococcus voltae Grown in the Presence of Mineral and Nonmineral Sources of Iron and Sulfur
(American Society for Microbiology, 2022-08) Steward, Katherine F.; Payne, Devon; Kincannon, Will; Johnson, Christina; Lensing, Malachi; Fausset, Hunter; Németh, Brigitta; Shepard, Eric M.; Broderick, William E.; Broderick, Joan B.; Dubois, Jen; Bothner, Brian
Clusters of iron and sulfur are key components of the active sites of enzymes that facilitate microbial conversion of light or electrical energy into chemical bonds. The proteins responsible for transporting iron and sulfur into cells and assembling these elements into metal clusters are not well understood.
Radical S -Adenosyl-l-methionine Chemistry in the Synthesis of Hydrogenase and Nitrogenase Metal Cofactors
(2014-12) Byer, Amanda S.; Shepard, Eric M.; Peters, John W.; Broderick, Joan B.
Nitrogenase, [FeFe]-hydrogenase, and [Fe]-hydrogenase enzymes perform catalysis at metal cofactors with biologically unusual non-protein ligands. The FeMo cofactor of nitrogenase has a MoFe7S9 cluster with a central carbon, whereas the H-cluster of [FeFe]-hydrogenase contains a 2Fe subcluster coordinated by cyanide and CO ligands as well as dithiomethylamine; the [Fe]-hydrogenase cofactor has CO and guanylylpyridinol ligands at a mononuclear iron site. Intriguingly, radical S-adenosyl-L-methionine enzymes are vital for the assembly of all three of these diverse cofactors. This minireview presents and discusses the current state of knowledge of the radical S-adenosylmethionine enzymes required for synthesis of these remarkable metal cofactors.