Setting an Upper Limit on the Myoglobin Iron(IV)Hydroxide p<i>K</i>_a: Insight into Axial Ligand Tuning in Heme Protein Catalysis

To provide insight into the iron­(IV)­hydroxide p<i>K</i>_a of histidine ligated heme proteins, we have probed the active site of myoglobin compound II over the pH range of 3.9–9.5, using EXAFS, Mössbauer, and resonance Raman spectroscopies. We find no indication of ferryl protonation over this pH range, allowing us to set an upper limit of 2.7 on the iron­(IV)­hydroxide p<i>K</i>_a in myoglobin. Together with the recent determination of an iron­(IV)­hydroxide p<i>K</i>_a ∼ 12 in the thiolate-ligated heme enzyme cytochrome P450, this result provides insight into Nature’s ability to tune catalytic function through its choice of axial ligand

A 2.8 Å Fe–Fe Separation in the Fe₂^III/IV Intermediate, X, from <i>Escherichia coli</i> Ribonucleotide Reductase

A class Ia ribonucleotide reductase (RNR) employs a μ-oxo-Fe₂^III/III/tyrosyl radical cofactor in its β subunit to oxidize a cysteine residue ∼35 Å away in its α subunit; the resultant cysteine radical initiates substrate reduction. During self-assembly of the <i>Escherichia coli</i> RNR-β cofactor, reaction of the protein’s Fe₂^II/II complex with O₂ results in accumulation of an Fe₂^III/IV cluster, termed <b>X</b>, which oxidizes the adjacent tyrosine (Y₁₂₂) to the radical (Y₁₂₂^•) as the cluster is converted to the μ-oxo-Fe₂^III/III product. As the first high-valent non-heme-iron enzyme complex to be identified and the key activating intermediate of class Ia RNRs, <b>X</b> has been the focus of intensive efforts to determine its structure. Initial characterization by extended X-ray absorption fine structure (EXAFS) spectroscopy yielded a Fe–Fe separation (<i>d</i>_Fe–Fe) of 2.5 Å, which was interpreted to imply the presence of three single-atom bridges (O^2–, HO^–, and/or μ-1,1-carboxylates). This short distance has been irreconcilable with computational and synthetic models, which all have <i>d</i>_Fe–Fe ≥ 2.7 Å. To resolve this conundrum, we revisited the EXAFS characterization of <b>X</b>. Assuming that samples containing increased concentrations of the intermediate would yield EXAFS data of improved quality, we applied our recently developed method of generating O₂ <i>in situ</i> from chlorite using the enzyme chlorite dismutase to prepare <b>X</b> at ∼2.0 mM, more than 2.5 times the concentration realized in the previous EXAFS study. The measured <i>d</i>_Fe–Fe = 2.78 Å is fully consistent with computational models containing a (μ-oxo)₂-Fe₂^III/IV core. Correction of the <i>d</i>_Fe–Fe brings the experimental data and computational models into full conformity and informs analysis of the mechanism by which <b>X</b> generates Y₁₂₂^•

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