Expression, purification, and activation of one key enzyme in anaerobic CO fixation: Carbon monoxide dehydrogenase II from Carboxydothermus hydrogenoformans.

Climate change due to anthropomorphic emissions will increase global temperature by at least 1.5 °C by the year 2030. One strategy to reduce the severity of the effects of climate change is to sequester carbon dioxide via natural biochemical cycles.

S-adenosyl-L-methionine is the unexpected methyl donor for the methylation of mercury by the membrane-associated HgcAB complex.

Mercury (Hg) is a heavy metal that exhibits high biological toxicity. Monomethylmercury and dimethylmercury are neurotoxins and a significant environmental concern as they bioaccumulate and biomagnify within the aquatic food web. Microbial Hg methylation involves two proteins, HgcA and HgcB.

Nature-Inspired Radical Pyridoxal-Mediated C-C Bond Formation.

Pyridoxal-5'-phosphate (PLP) and derivatives of this cofactor enable a plethora of reactions in both enzyme-mediated and free-in-solution transformations. With few exceptions in each category, such chemistry has predominantly involved two-electron processes. This sometimes poses a significant challenge for using PLP to build tetrasubstituted carbon centers, especially when the reaction is reversible.

An alcove at the acetyl-CoA synthase nickel active site is required for productive substrate CO binding and anaerobic carbon fixation.

One of the seven natural CO fixation pathways, the anaerobic Wood-Ljungdahl pathway (WLP) is unique in generating CO as a metabolic intermediate, operating through organometallic intermediates, and in conserving (versus utilizing) net ATP. The key enzyme in the WLP is acetyl-CoA synthase (ACS), which uses an active site [2Ni-4Fe-4S] cluster (A-cluster), a CO tunnel, and an organometallic (Ni-CO, Ni-methyl, and Ni-acetyl) reaction sequence to generate acetyl-CoA. Here, we reveal that an alcove, which interfaces the tunnel and the A-cluster, is essential for CO fixation and autotrophic growth by the WLP.

Characterization of Methyl- and Acetyl-Ni Intermediates in Acetyl CoA Synthase Formed during Anaerobic CO and CO Fixation.

The Wood-Ljungdahl Pathway is a unique biological mechanism of carbon dioxide and carbon monoxide fixation proposed to operate through nickel-based organometallic intermediates. The most unusual steps in this metabolic cycle involve a complex of two distinct nickel-iron-sulfur proteins: CO dehydrogenase and acetyl-CoA synthase (CODH/ACS). Here, we describe the nickel-methyl and nickel-acetyl intermediates in ACS completing the characterization of all its proposed organometallic intermediates.

Heme delivery to heme oxygenase-2 involves glyceraldehyde-3-phosphate dehydrogenase.

Heme regulatory motifs (HRMs) are found in a variety of proteins with diverse biological functions. In heme oxygenase-2 (HO2), heme binds to the HRMs and is readily transferred to the catalytic site in the core of the protein. To further define this heme transfer mechanism, we evaluated the ability of GAPDH, a known heme chaperone, to transfer heme to the HRMs and/or the catalytic core of HO2.

Efficient, Light-Driven Reduction of CO to CO by a Carbon Monoxide Dehydrogenase-CdSe/CdS Nanorod Photosystem.

The solar conversion of CO to low carbon fuels has been heralded as a potential solution to combat the rise in greenhouse gas emissions. Here we report the first light-driven activation of [NiFe] CODH II from for the reduction of CO to CO. To accomplish this, a hybrid photosystem composed of CODH II and CdSe/CdS dot-in-rod nanocrystals was developed.

XFEL serial crystallography reveals the room temperature structure of methyl-coenzyme M reductase.

Methyl-Coenzyme M Reductase (MCR) catalyzes the biosynthesis of methane in methanogenic archaea, using a catalytic Ni-centered Cofactor F430 in its active site. It also catalyzes the reverse reaction, that is, the anaerobic activation and oxidation, including the cleavage of the CH bond in methane. Because methanogenesis is the major source of methane on earth, understanding the reaction mechanism of this enzyme can have massive implications in global energy balances.

Heme oxygenase-2 (HO-2) binds and buffers labile ferric heme in human embryonic kidney cells.

Heme oxygenases (HOs) detoxify heme by oxidatively degrading it into carbon monoxide, iron, and biliverdin, which is reduced to bilirubin and excreted. Humans express two isoforms of HO: the inducible HO-1, which is upregulated in response to excess heme and other stressors, and the constitutive HO-2. Much is known about the regulation and physiological function of HO-1, whereas comparatively little is known about the role of HO-2 in regulating heme homeostasis.

Not a "they" but a "we": The microbiome helps promote our well-being.

Anaerobic microbes in the human gut produce beneficial and harmful compounds, as well as neutral compounds like trimethylamine, which undergoes microbial metabolism or host-catalyzed transformation into proatherogenic trimethylamine-N-oxide. Ellenbogen et al. identified a microbiome-associated demethylase that short-circuits the production of trimethylamine-N-oxide from the metabolite γ-butyrobetaine and instead produces methyltetrahydrofolate, a key intermediate in the microbial production of beneficial small-chain fatty acids.

Regulation of protein function and degradation by heme, heme responsive motifs, and CO.

Heme is an essential biomolecule and cofactor involved in a myriad of biological processes. In this review, we focus on how heme binding to heme regulatory motifs (HRMs), catalytic sites, and gas signaling molecules as well as how changes in the heme redox state regulate protein structure, function, and degradation. We also relate these heme-dependent changes to the affected metabolic processes.

Nickel-Sulfonate Mode of Substrate Binding for Forward and Reverse Reactions of Methyl-SCoM Reductase Suggest a Radical Mechanism Involving Long-Range Electron Transfer.

Methyl-coenzyme M reductase (MCR) catalyzes both the synthesis and the anaerobic oxidation of methane (AOM). Its catalytic site contains Ni at the core of cofactor F. The Ni ion, in its low-valent Ni(I) state, lights the fuse leading to homolysis of the C-S bond of methyl-coenzyme M (methyl-SCoM) to generate a methyl radical, which abstracts a hydrogen atom from coenzyme B (HSCoB) to generate methane and the mixed disulfide CoMSSCoB.

Crystallographic Characterization of the Carbonylated A-Cluster in Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase.

The Wood-Ljungdahl pathway allows for autotrophic bacterial growth on carbon dioxide, with the last step in acetyl-CoA synthesis catalyzed by the bifunctional enzyme carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS). ACS uses a complex Ni-Fe-S metallocluster termed the A-cluster to assemble acetyl-CoA from carbon monoxide, a methyl moiety and coenzyme A. Here, we report the crystal structure of CODH/ACS from with substrate carbon monoxide bound at the A-cluster, a state previously uncharacterized by crystallography.

Ferric heme as a CO/NO sensor in the nuclear receptor Rev-Erbß by coupling gas binding to electron transfer.

Rev-Erbβ is a nuclear receptor that couples circadian rhythm, metabolism, and inflammation. Heme binding to the protein modulates its function as a repressor, its stability, its ability to bind other proteins, and its activity in gas sensing. Rev-Erbβ binds Fe-heme more tightly than Fe-heme, suggesting its activities may be regulated by the heme redox state.

Heme oxygenase-2 is post-translationally regulated by heme occupancy in the catalytic site.

Heme oxygenase-2 (HO2) and -1 (HO1) catalyze heme degradation to biliverdin, CO, and iron, forming an essential link in the heme metabolism network. Tight regulation of the cellular levels and catalytic activities of HO1 and HO2 is important for maintaining heme homeostasis. HO1 expression is transcriptionally regulated; however, HO2 expression is constitutive.

Negative-Stain Electron Microscopy Reveals Dramatic Structural Rearrangements in Ni-Fe-S-Dependent Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase.

The Ni-Fe-S-containing A-cluster of acetyl-coenzyme A (CoA) synthase (ACS) assembles acetyl-CoA from carbon monoxide (CO), a methyl group (CH), and CoA. To accomplish this feat, ACS must bind CoA and interact with two other proteins that contribute the CO and CH, respectively: CO dehydrogenase (CODH) and corrinoid Fe-S protein (CFeSP). Previous structural data show that, in the model acetogen Moorella thermoacetica, domain 1 of ACS binds to CODH such that a 70-Å-long internal channel is created that allows CO to travel from CODH to the A-cluster.

C Electron Nuclear Double Resonance Spectroscopy Shows Acetyl-CoA Synthase Binds Two Substrate CO in Multiple Binding Modes and Reveals the Importance of a CO-Binding "Alcove".

EPR and Electron Nuclear Double Resonance spectroscopies here characterize CO binding to the active-site A cluster of wild-type (WT) Acetyl-CoA Synthase (ACS) and two variants, F229W and F229A. The A-cluster binds CO to a proximal Ni (Ni) that bridges a [4Fe-4S] cluster and a distal Ni. An alcove seen in the ACS crystal structure near the A-cluster, defined by hydrophobic residues including F229, forms a cage surrounding a Xe mimic of CO.

Structure determination of the HgcAB complex using metagenome sequence data: insights into microbial mercury methylation.

Bacteria and archaea possessing the hgcAB gene pair methylate inorganic mercury (Hg) to form highly toxic methylmercury. HgcA consists of a corrinoid binding domain and a transmembrane domain, and HgcB is a dicluster ferredoxin. However, their detailed structure and function have not been thoroughly characterized.

The heme-regulatory motifs of heme oxygenase-2 contribute to the transfer of heme to the catalytic site for degradation.

Heme-regulatory motifs (HRMs) are present in many proteins that are involved in diverse biological functions. The C-terminal tail region of human heme oxygenase-2 (HO2) contains two HRMs whose cysteine residues form a disulfide bond; when reduced, these cysteines are available to bind Fe-heme. Heme binding to the HRMs occurs independently of the HO2 catalytic active site in the core of the protein, where heme binds with high affinity and is degraded to biliverdin.

Oxygen and Conformation Dependent Protein Oxidation and Aggregation by Porphyrins in Hepatocytes and Light-Exposed Cells.

Background & Aims: Porphyrias are caused by porphyrin accumulation resulting from defects in the heme biosynthetic pathway that typically lead to photosensitivity and possible end-stage liver disease with an increased risk of hepatocellular carcinoma. Our aims were to study the mechanism of porphyrin-induced cell damage and protein aggregation, including liver injury, where light exposure is absent.

Methods: Porphyria was induced in vivo in mice using 3,5-diethoxycarbonyl-1,4-dihydrocollidine or in vitro by exposing human liver Huh7 cells and keratinocytes, or their lysates, to protoporphyrin-IX, other porphyrins, or to δ-aminolevulinic acid plus deferoxamine.

Kinetics of Enzymatic Mercury Methylation at Nanomolar Concentrations Catalyzed by HgcAB.

Methylmercury (MeHg) is a potent bioaccumulative neurotoxin that is produced by certain anaerobic bacteria and archaea. Mercury (Hg) methylation has been linked to the gene pair , which encodes a membrane-associated corrinoid protein and a ferredoxin. Although microbial Hg methylation has been characterized , the cellular biochemistry and the specific roles of the gene products HgcA and HgcB in Hg methylation are not well understood.

Dynamic and structural differences between heme oxygenase-1 and -2 are due to differences in their C-terminal regions.

Heme oxygenase (HO) catalyzes heme degradation, a process crucial for regulating cellular levels of this vital, but cytotoxic, cofactor. Two HO isoforms, HO1 and HO2, exhibit similar catalytic mechanisms and efficiencies. They also share catalytic core structures, including the heme-binding site.

Production and properties of enzymes that activate and produce carbon monoxide.

The chapter focuses on the methods involved in producing and characterizing two key nickel-iron-sulfur enzymes in the Wood-Ljungdahl pathway (WLP) of anaerobic conversion of carbon dioxide fixation into acetyl-CoA: carbon monoxide dehydrogenase (CODH) and acetyl-CoA synthase (ACS). The WLP is used for biosynthesis of cell material and energy conservation by anaerobic bacteria and archaea, and it is central to several industrial biotechnology processes aimed at using syngas and waste gases for the production of fuels and chemicals. The pathway can run in reverse to allow organisms, e.

An unlikely heme chaperone confirmed at last.

Labile heme, as opposed to heme that is tightly bound within proteins, is thought to require a chaperone to be trafficked within the cell due to its cytotoxicity, but the identity of this chaperone was not known. A new study reveals that an unlikely protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is a heme chaperone that binds and transfers labile heme to downstream target proteins. These results provide a new framework for understanding heme homeostasis and raise intriguing questions regarding the intersection of heme transport, carbohydrate metabolism, and intracellular signaling.