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Peptidylglycine monooxygenase (PHM) is essential for the posttranslational amidation of neuroendocrine peptides. An important aspect of the PHM mechanism is the complete coupling of oxygen reduction to substrate hydroxylation, which implies no oxygen reactivity of the fully reduced enzyme in the absence of peptidyl substrates. As part of studies aimed at investigating this feature of the PHM mechanism, we explored pre-steady-state kinetics using chemical quench (CQ) and rapid freeze-quench (RFQ) studies of the fully reduced ascorbate-free PHM enzyme. First, we confirmed the absence of Cu(I)-enzyme oxidation by O

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. We modeled the HgcAB complex by combining metagenome sequence data mining, coevolution analysis, and Rosetta structure calculations. In addition, we overexpressed HgcA and HgcB in Escherichia coli, confirmed spectroscopically that they bind cobalamin and [4Fe-4S] clusters, respectively, and incorporated these cofactors into the structural model. Surprisingly, the two domains of HgcA do not interact with each other, but HgcB forms extensive contacts with both domains. The model suggests that conserved cysteines in HgcB are involved in shuttling HgII, methylmercury, or both. These findings refine our understanding of the mechanism of Hg methylation and expand the known repertoire of corrinoid methyltransferases in nature., Connor J. Cooper et al. expressed HgcA and HgcB in Escherichia coli and modeled the structure of the HgcAB complex by combining metagenome sequence data, coevolution analysis, and ab initio structure calculations. This study provides insights into the biochemical mechanism of mercury (Hg) methylation.

Mucus protects the epithelial cells of the digestive and respiratory tracts from pathogens and other hazards. Progress in determining the molecular mechanisms of mucus barrier function has been limited by the lack of high-resolution structural information on mucins, the giant, secreted, gel-forming glycoproteins that are the major constituents of mucus. Here, we report how mucin structures we determined enabled the discovery of an unanticipated protective role of mucus: managing the toxic transition metal copper. Using two juxtaposed copper binding sites, one for Cu

SUMMARYMucus protects the body by many mechanisms, but a role in managing toxic transition metals was not previously known. Here we report that secreted mucins, the major mucus glycoproteins coating the respiratory and intestinal epithelia, are specific copper-binding proteins. Most remarkably, the intestinal mucin, MUC2, has two juxtaposed copper binding sites, one that accommodates Cu2+and the other Cu1+, which can be formedin situby reduction with vitamin C. Copper is an essential trace metal because it is a cofactor for a variety of enzymes catalyzing electron transfer reactions, but copper damages macromolecules when unregulated. We observed that MUC2 protects against copper toxicity while permitting nutritional uptake into cells. These findings introduce mucins, produced in massive quantities to guard extensive mucosal surfaces, as extracellular copper chaperones and potentially important players in physiological copper homeostasis.

An important question is whether consensus mechanisms for copper monooxygenase enzymes such as peptidylglycine monooxygenase (PHM) and dopamine β-monooxygenase (DBM) generated via computational and spectroscopic approaches account for important experimental observations. We examine this question in the light of recent crystallographic and QMMM reports which suggest that alternative mechanisms involving an open to closed conformational cycle may be more representative of a number of experimental findings that remain unaccounted for in the canonical mononuclear mechanisms. These include (i) the almost negligible reactivity of the catalytic copper site (CuM) with oxygen in the absence of substrate, (ii) the carbonyl chemistry and in particular the substrate-induced activation exemplified by the lowered CO stretching frequency, (iii) the peroxide shunt chemistry which demands an intermediate that facilitates equilibrium between a Cu(II)-peroxo state and a Cu(I)-dioxygen state, and (iv) clear evidence for both closed and open conformational states in both PHM and DBM. An alternative mechanism involving a dinuclear copper intermediate formed via an open to closed conformational transition appears better able to accommodate these experimental observations, as well as being shown by QMMM methodologies to be energetically feasible. This suggests that future experiments should be designed to distinguish between these competing mechanisms and the factors that govern the oxygen reactivity of the copper centers. In particular, determining how oxygen reactivity is activated by binding of substrate, should be considered an important new challenge.