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Abstract: Horseradish peroxidase (HRP), isolated from horseradish roots, is heavily glycosylated, making it difficult to crystallize. In this work, we produced recombinant HRP in E. coli and obtained an X-ray structure of the ferric enzyme at 1.63 Å resolution. The structure shows that the recombinant HRP contains four disulphide bonds and two calcium ions, which are highly conserved in class III peroxidase enzymes. The heme active site contains histidine residues at the proximal (His 170) and distal (His 42) positions, and an active site arginine (Arg 38). Surprisingly, an ethylene glycol molecule was identified in the active site, forming hydrogen bonds with His 42 and Arg 38 at the δ-heme edge. The high yields obtained from the recombinant expression system, and the successful crystallization of the enzyme pave the way for new structural studies in the future.

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Abstract: Campylobacter jejuni is a pathogenic bacteria that causes gastrointestinal disorders and is thus of great importance. Phosphorylating Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a ubiquitous cellular enzyme that has a well-defined role in glycolysis and other pathways where it catalyses the oxidative phosphorylation of glyceraldehyde 3-phosphate (2-hydroxy-3-oxopropyl dihydrogen phosphate) to 1,3-Bisphosphoglycerate ((2-Hydroxy-3-phosphonooxy-propanoyloxy)phosphonic acid). The C. jejuni genome encodes a single GAPDH enzyme (CjGAPDH) which displays dual (NAD/NADP) coenzyme specificity. NAD-specific GAPDHs are given the EC classification of 1.2.1.12, whereas NADP-specific GAPDHs are classed as 1.2.1.13. GAPDH's with dual specificity are in the class 1.2.1.59. Here we present the X-ray crystal structure of this enzyme (at 2.25 Å), this comprises superimposed structures of NAD- and NADP- complexes showing the structural adaptation that allows this dual specificity, and we consider this in the context of the pathogen's metabolism. There are no previous reports of EC 1.2.1.59 structures that compare the binding of the two co-enzymes. Furthermore, we also report the structure (at 2.05 Å) of the enzyme complexed with the nucleoside ADP and consider this with respect to the reported “moonlighting” activities of GAPDH.

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Abstract: The ether-à-go-go (EAG) family of voltage gated K+ channels are important regulators of neuronal and cardiac action potential firing (excitability) and have major roles in human diseases such as epilepsy, schizophrenia, cancer and sudden cardiac death. A defining feature of EAG (Kv10-12) channels is a highly conserved domain on the amino-terminus, known as the eag-domain, consisting of a PAS domain capped by a short sequence containing an amphipathic helix (Cap-domain). The PAS and Cap domains are both vital for the normal function of EAG channels. Using heme-affinity pull-down assays and proteomics of lysates from primary cortical neurons, we identified that an EAG channel, hERG3 (Kv11.3), binds to heme. In whole cell electrophysiology experiments, we identified that heme inhibits hERG3 channel activity. In addition, we expressed the Cap and PAS domain of hERG3 in E.coli and, using spectroscopy and kinetics, identified the PAS domain as the location for heme binding. The results identify heme as a regulator of hERG3 channel activity. These observations are discussed in the context of the emerging role for heme as a regulator of ion channel activity in cells.

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Abstract: Aerobic organisms have evolved to activate oxygen from the atmosphere, which allows them to catalyze the oxidation of different kinds of substrates. This activation of oxygen is achieved by a metal center (usually iron or copper) buried within a metalloprotein. In the case of iron-containing heme enzymes, the activation of oxygen is achieved by formation of transient iron-oxo (ferryl) intermediates; these intermediates are called Compound I and Compound II. The Compound I and II intermediates were first discovered in the 1930s in horseradish peroxidase, and it is now known that these same species are used across the family of heme enzymes, which include all of the peroxidases, the heme catalases, the P450s, cytochrome c oxidase, and NO synthase. Many years have passed since the first observations, but establishing the chemical nature of these transient ferryl species remains a fundamental question that is relevant to the reactivity, and therefore the usefulness, of these species in biology. This Account summarizes experiments that were conceived and conducted at Leicester and presents our ideas on the chemical nature, stability, and reactivity of these ferryl heme species. We begin by briefly summarizing the early milestones in the field, from the 1940s and 1950s. We present comparisons between the nature and reactivity of the ferryl species in horseradish peroxidase, cytochrome c peroxidase, and ascorbate peroxidase; and we consider different modes of electron delivery to ferryl heme, from different substrates in different peroxidases. We address the question of whether the ferryl heme is best formulated as an (unprotonated) FeIV═O or as a (protonated) FeIV–OH species. A range of spectroscopic approaches (EXAFS, resonance Raman, Mossbauer, and EPR) have been used over many decades to examine this question, and in the last ten years, X-ray crystallography has also been employed. We describe how information from all of these studies has blended together to create an overall picture, and how the recent application of neutron crystallography has directly identified protonation states and has helped to clarify the precise nature of the ferryl heme in cytochrome c peroxidase and ascorbate peroxidase. We draw comparisons between the Compound I and Compound II species that we have observed in peroxidases with those found in other heme systems, notably the P450s, highlighting possible commonality across these heme ferryl systems. The identification of proton locations from neutron structures of these ferryl species opens the door for understanding the proton translocations that need to occur during O–O bond cleavage.

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Abstract: Collectin-K1 (CL-K1, or CL-11) is a multifunctional Ca2+-dependent lectin with roles in innate immunity, apoptosis and embryogenesis. It binds to carbohydrates on pathogens to activate the lectin pathway of complement and together with its associated serine protease MASP-3 serves as a guidance cue for neural crest development. High serum levels are associated with disseminated intravascular coagulation, where spontaneous clotting can lead to multiple organ failure. Autosomal mutations in the CL-K1 or MASP-3 genes cause a developmental disorder called 3MC (Carnevale, Mingarelli, Malpuech and Michels) syndrome, characterised by facial, genital, renal and limb abnormalities. One of these mutations (Gly204Ser in the CL-K1 gene) is associated with undetectable levels of protein in the serum of affected individuals.