DNA charge transport chemistry offers a means of long range, rapid redox signaling. Here we demonstrate that the [4Fe4S] cluster in human DNA primase can utilize this chemistry to coordinate the first steps of replication. Through DNA electrochemistry, we find that a change in oxidation state of the [4Fe4S] cluster acts as a switch for DNA binding. Single-atom mutations that inhibit this charge transfer, moreover, hinder primase initiation without affecting primase structure or polymerization. Generating a single base mismatch in the growing primer duplex, which attenuates DNA charge transport, inhibits primer truncation. Thus redox signaling by [4Fe4S] clusters using DNA charge transport regulates primase binding to DNA and illustrates chemistry that may efficiently drive substrate handoff between polymerases during DNA replication.

Dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a bifunctional enzyme that possesses both hemoglobin and peroxidase activities. Of the two DHP isoenzymes identified to date, much of the recent focus has been on DHP A, whereas very little is known pertaining to the activity, substrate specificity, mechanism of function, or spectroscopic properties of DHP B. Herein, we report the recombinant expression and purification of DHP B, as well as the details of our investigations into its catalytic cycle using biochemical assays, stopped-flow UVvisible, resonance Raman and rapid-freeze-quench electron paramagnetic resonance spectroscopies, and spectroelectrochemistry. Our experimental design reveals mechanistic insights and kinetic descriptions of the dehaloperoxidase mechanism which have not been previously reported for isoenzyme A. Namely, we demonstrate a novel reaction pathway in which the products of the oxidative dehalogenation of trihalophenols (dihaloquinones) are themselves capable of inducing formation of oxyferrous DHP B, and an updated catalytic cycle for DHP is proposed. We further demonstrate that unlike the traditional monofunctional peroxidases, the oxyferrous state in DHP is a peroxidase competent starting species, which suggests that the ferric oxidation state may not be an obligatory starting point for the enzyme. The data presented herein provide a link between the peroxidase and oxygen transport activities which furthers our understanding of how this bifunctional enzyme is able to unite its two inherent functions in one system. Figure SD1); UVvisible spectra of the (tri)halophenol complexes of DHP B ( Figure SD2); UV-visible spectroscopic monitoring of the oxidative dehalogenation of trihalophenols as catalyzed by DHP B in the presence of hydrogen peroxide ( Figure SD3); dependence of k obs for the reaction between ferric DHP B with hydrogen peroxide (2.5 -25 equivalents) at pH 7 yielding Compound ES ( Figure SD4); stopped-flow UV-visible spectroscopic monitoring of ( Figure SD5), and DCQ product formation and TCP co-substrate loss for ( Figure SD6), the double-mixing reaction between preformed DHP B Compound ES and TCP at pH 7; stopped-flow UV-visible spectroscopic monitoring of ( Figure SD7), and DCQ product formation and TCP co-substrate loss for ( Figure SD8), the doublemixing reaction between ferric DHP B pre-incubated with TCP for 500 ms prior to its reaction with a 10-fold excess of H 2 O 2 (in situ generated Compound ES) at pH 7; stopped-flow UV-visible spectroscopic monitoring of the double-mixing reaction between ferric DHP B pre-incubated with a 7-fold molar excess of DCQ for 500 ms prior to its reaction with a 2.5-fold excess of H 2 O 2 (in situ generated Compound ES) ( Figure SD9); stopped-flow UV-visible spectroscopic monitoring of the reaction between ferric DHP B and a 7-fold excess of DCQ at pH 7 ( Figure SD10); reduction of Compound RH yielding Compound P 426 ( Figure SD11), reduction of Compound RH yielding oxyferrous DHP B ( Figure SD12). This m...

Dehaloperoxidase (DHP) from the annelid Amphitrite ornata is a catalytically active hemoglobin-peroxidase that possesses a unique internal binding cavity in the distal pocket above the heme. The previously published crystal structure of DHP shows 4-iodophenol bound internally. This led to the proposal that the internal binding site is the active site for phenol oxidation. However, the native substrate for DHP is 2,4,6-tribromophenol, and all attempts to bind 2,4,6-tribromophenol in the internal site under physiological conditions have failed. Herein, we show that the binding of 4-halophenols in the internal pocket inhibits enzymatic function. Furthermore, we demonstrate that DHP has a unique two-site competitive binding mechanism in which the internal and external binding sites communicate through two conformations of the distal histidine of the enzyme, resulting in nonclassical competitive inhibition. The same distal histidine conformations involved in DHP function regulate oxygen binding and release during transport and storage by hemoglobins and myoglobins. This work provides further support for the hypothesis that DHP possesses an external binding site for substrate oxidation, as is typical for the peroxidase family of enzymes.

The fosfomycin resistance enzymes, FosB, from Gram-positive organisms, are M2+ dependent thiol tranferases that catalyze nucleophilic addition of either L-cysteine (L-cys) or bacillithiol (BSH) to the antibiotic, resulting in a modified compound with no bacteriacidal properties. Here we report the structural and functional characterization of FosB from Bacillus cereus (FosBBc). The overall structure of FosBBc, at 1.27 Å resolution, reveals that the enzyme belongs to the vicinal oxygen chelate (VOC) superfamily. Crystal structures of FosBBc co-crystallized with fosfomycin and a variety of divalent metals, including Ni2+, Mn2+, Co2+, and Zn2+, indicate that the antibiotic coordinates to the active site metal center in an orientation similar to that found in the structurally homologous manganese-dependent fosfomycin resistance enzyme, FosA. Surface analysis of the FosBBc structures show a well-defined binding pocket and an access channel to C1 of fosfomycin, the carbon to which nucleophilic addition of the thiol occurs. The pocket and access channel are appropriate in size and shape to accommodate L-cys or BSH. Further investigation of the structures revealed that the fosfomycin molecule, anchored by the metal, is surrounded by a cage of amino acids that hold the antibiotic in an orientation such that C1 is centered at the end of the solvent channel positioning the compound for direct nucleophilic attack by the thiol substrate. In addition, the structures of FosBBc in complex with the L-cysteine-fosfomycin product (1.55 Å resolution) and in complex with the bacillithiol-fosfomycin product (1.77 Å resolution) coordinated to a Mn2+ metal in the active site have been determined. The L-cysteine moiety of either product is located in the solvent channel, where the thiol has added to the backside of fosfomycin C1 located at the end of the channel. Concomitant kinetic analyses of FosBBc indicated that the enzyme has a preference for bacillithiol over L-cysteine when activated by Mn2+ and is inhibited by Zn2+. The fact that Zn2+ is an inhibitor of FosBBc was used to obtain a ternary complex structure of the enzyme with both fosfomycin and L-cysteine bound.

The present work highlights the important role played by the distal histidine in controlling the binding of heme ligands in dehaloperoxidase (DHP) as compared to myoglobin and peroxidases. In DHP the distal histidine is highly mobile and undergoes a conformational change that places it within hydrogen-bonding distance of anionic ligands and water, where strong hydrogen bonding can occur. The detailed resonance Raman (RR) analysis at room temperature shows the presence of an equilibrium between a 5-coordinate and a 6-coordinate (aquo) high-spin form. The equilibrium shifts toward the aquo form at 12 K. These two forms are consistent with the existing X-ray structures where a closed conformation, with His55 positioned in the distal pocket and H-bonded with the distal water molecule (6-coordinate), and an open solvent-exposed conformation, with the His55 displaced from the distal pocket (5-coordinate form), are in equilibrium. Moreover, the comparison between the Raman data at 298 and 12 K and the results obtained by EPR of DHP in the presence of 4-iodophenol highlights the formation of a pure 5-coordinate high-spin form (open conformation). The data reported herein support the role of His55 in facilitating the interaction of substrate and inhibitor in the regulation of enzyme function, as previously suggested. The two conformations of His55 in equilibrium at room temperature provide a level of control that permits the distal histidine to act as both the acid-base catalyst in the peroxidase mechanism and the stabilizing amino acid for exogenous heme-coordinated ligands.