Direct Measurement of the Radical Translocation Distance in the Class I Ribonucleotide Reductase from <i>Chlamydia trachomatis</i>

Ribonucleotide reductases (RNRs) catalyze conversion of ribonucleotides to deoxyribonucleotides in all organisms via a free-radical mechanism that is essentially conserved. In class I RNRs, the reaction is initiated and terminated by radical translocation (RT) between the α and β subunits. In the class Ic RNR from <i>Chlamydia trachomatis</i> (<i>Ct</i> RNR), the initiating event converts the active <i>S</i> = 1 Mn­(IV)/Fe­(III) cofactor to the <i>S</i> = 1/2 Mn(III)/Fe(III) “RT-product” form in the β subunit and generates a cysteinyl radical in the α active site. The radical can be trapped via the well-described decomposition reaction of the mechanism-based inactivator, 2′-azido-2′-deoxyuridine-5′-diphosphate, resulting in the generation of a long-lived, nitrogen-centered radical (N^•) in α. In this work, we have determined the distance between the Mn­(III)/Fe­(III) cofactor in β and N^• in α to be 43 ± 1 Å by using double electron–electron resonance experiments. This study provides the first structural data on the <i>Ct</i> RNR holoenzyme complex and the first direct experimental measurement of the inter-subunit RT distance in any class I RNR

Experimental Correlation of Substrate Position with Reaction Outcome in the Aliphatic Halogenase, SyrB2

The iron­(II)- and 2-(oxo)­glutarate-dependent (Fe/2OG) oxygenases catalyze an array of challenging transformations, but how individual members of the enzyme family direct different outcomes is poorly understood. The Fe/2OG halogenase, SyrB2, chlorinates C4 of its native substrate, l-threonine appended to the carrier protein, SyrB1, but hydroxylates C5 of l-norvaline and, to a lesser extent, C4 of l-aminobutyric acid when SyrB1 presents these non-native amino acids. To test the hypothesis that positioning of the targeted carbon dictates the outcome, we defined the positions of these three substrates by measuring hyperfine couplings between substrate deuterium atoms and the stable, EPR-active iron–nitrosyl adduct, a surrogate for reaction intermediates. The Fe–²H distances and N–Fe–²H angles, which vary from 4.2 Å and 85° for threonine to 3.4 Å and 65° for norvaline, rationalize the trends in reactivity. This experimental correlation of position to outcome should aid in judging from structural data on other Fe/2OG enzymes whether they suppress hydroxylation or form hydroxylated intermediates on the pathways to other outcomes

Spectroscopic Investigations of Catalase Compound II: Characterization of an Iron(IV) Hydroxide Intermediate in a Non-thiolate-Ligated Heme Enzyme

We report on the protonation state of <i>Helicobacter pylori</i> catalase compound II. UV/visible, Mössbauer, and X-ray absorption spectroscopies have been used to examine the intermediate from pH 5 to 14. We have determined that HPC-II exists in an iron­(IV) hydroxide state up to pH 11. Above this pH, the iron­(IV) hydroxide complex transitions to a new species (p<i>K</i>_a = 13.1) with Mössbauer parameters that are indicative of an iron­(IV)-oxo intermediate. Recently, we discussed a role for an elevated compound II p<i>K</i>_a in diminishing the compound I reduction potential. This has the effect of shifting the thermodynamic landscape toward the two-electron chemistry that is critical for catalase function. In catalase, a diminished potential would increase the selectivity for peroxide disproportionation over off-pathway one-electron chemistry, reducing the buildup of the inactive compound II state and reducing the need for energetically expensive electron donor molecules

Function of the Diiron Cluster of <i>Escherichia coli</i> Class Ia Ribonucleotide Reductase in Proton-Coupled Electron Transfer

The class Ia ribonucleotide reductase (RNR) from <i>Escherichia coli</i> employs a free-radical mechanism, which involves bidirectional translocation of a radical equivalent or “hole” over a distance of ∼35 Å from the stable diferric/tyrosyl-radical (Y₁₂₂^•) cofactor in the β subunit to cysteine 439 (C₄₃₉) in the active site of the α subunit. This long-range, intersubunit electron transfer occurs by a multistep “hopping” mechanism via formation of transient amino acid radicals along a specific pathway and is thought to be conformationally gated and coupled to local proton transfers. Whereas constituent amino acids of the hopping pathway have been identified, details of the proton-transfer steps and conformational gating within the β sununit have remained obscure; specific proton couples have been proposed, but no direct evidence has been provided. In the key first step, the reduction of Y₁₂₂^• by the first residue in the hopping pathway, a water ligand to Fe₁ of the diferric cluster was suggested to donate a proton to yield the neutral Y₁₂₂. Here we show that forward radical translocation is associated with perturbation of the Mössbauer spectrum of the diferric cluster, especially the quadrupole doublet associated with Fe₁. Density functional theory (DFT) calculations verify the consistency of the experimentally observed perturbation with that expected for deprotonation of the Fe₁-coordinated water ligand. The results thus provide the first evidence that the diiron cluster of this prototypical class Ia RNR functions not only in its well-known role as generator of the enzyme’s essential Y₁₂₂^•, but also directly in catalysis