Rachel K. Behan

Iron(IV)hydroxide pKa and the Role of Thiolate Ligation in C–H Bond Activation by Cytochrome P450

The pKa of P450 Cytochrome P450 enzymes oxidize hydrocarbons through activation of oxygen at heme iron centers. However, the protein backbone has various sites (particularly tyrosine residues) that are also sensitive to oxidation, so how can the enzyme rapidly transform substrates without attacking itself? Yosca et al. (p. 825) explored the energetics of the competition between substrate and self-oxidation by measuring the pKa of the enzymes iron(IV)hydroxide motif. Cysteine thiolate coordination to iron in the P450 structure raised the pKa almost to 12—rendering the iron oxo far more basic than analogous motifs in other heme environments. Correspondingly, the electronic environment for H-atom transfer from the substrate was relatively favorable, compared to electron transfer from a backbone residue. The basicity of an iron oxo intermediate helps explain what keeps P450 enzymes from oxidizing their own backbone. Cytochrome P450 enzymes activate oxygen at heme iron centers to oxidize relatively inert substrate carbon-hydrogen bonds. Cysteine thiolate coordination to iron is posited to increase the pKa (where Ka is the acid dissociation constant) of compound II, an iron(IV)hydroxide complex, correspondingly lowering the one-electron reduction potential of compound I, the active catalytic intermediate, and decreasing the driving force for deleterious auto-oxidation of tyrosine and tryptophan residues in the enzyme’s framework. Here, we report on the preparation of an iron(IV)hydroxide complex in a P450 enzyme (CYP158) in ≥90% yield. Using rapid mixing technologies in conjunction with Mössbauer, ultraviolet/visible, and x-ray absorption spectroscopies, we determine a pKa value for this compound of 11.9. Marcus theory analysis indicates that this elevated pKa results in a >10,000-fold reduction in the rate constant for oxidations of the protein framework, making these processes noncompetitive with substrate oxidation.

On the feasibility of N2 fixation via a single-site FeI/FeIV cycle: Spectroscopic studies of FeI(N2)FeI, FeIV N, and related species

The electronic properties of an unusually redox-rich iron system, [PhBPR3]FeNx (where [PhBPR3] is [PhB(CH2PR2)3]−), are explored by Mössbauer, EPR, magnetization, and density-functional methods to gain a detailed picture regarding their oxidation states and electronic structures. The complexes of primary interest in this article are the two terminal iron(IV) nitride species, [PhBPiPr3]FeN (3a) and [PhBPCH2Cy3]FeN (3b), and the formally diiron(I) bridged-Fe(μ-N2)Fe species, {[PhBPiPr3]Fe}2(μ-N2) (4). Complex 4 is chemically related to 3a via a spontaneous nitride coupling reaction. The diamagnetic iron(IV) nitrides 3a and 3b exhibit unique electronic environments that are reflected in their unusual Mössbauer parameters, including quadrupole-splitting values of 6.01(1) mm/s and isomer shift values of −0.34(1) mm/s. The data for 4 suggest that this complex can be described by a weak ferromagnetic interaction (J/D < 1) between two iron(I) centers. For comparison, four other relevant complexes also are characterized: a diamagnetic iron(IV) trihydride [PhBPiPr3]Fe(H)3(PMe3) (5), an S = 3/2 iron(I) phosphine adduct [PhBPiPr3]FePMe3 (6), and the S = 2 iron(II) precursors to 3a, [PhBPiPr3]FeCl and [PhBPiPr3]Fe-2,3:5,6-dibenzo-7-aza bicyclo[2.2.1]hepta-2,5-diene (dbabh). The electronic properties of these respective complexes also have been explored by density-functional methods to help corroborate our spectral assignments and to probe their electronic structures further.

Resonance Raman spectroscopy of chloroperoxidase compound II provides direct evidence for the existence of an iron(IV)-hydroxide

We report direct evidence for the existence of an iron(IV)–hydroxide. Resonance Raman measurements on chloroperoxidase compound II (CPO-II) reveal an isotope (18O and 2H)-sensitive band at νFe–O = 565 cm−1. Preparation of CPO-II in H2O using H218O2 results in a red-shift of 22 cm−1, while preparation of CPO-II in 2H2O using H2O2 results in a red-shift of 13 cm−1. These values are in good agreement with the isotopic shifts predicted (23 and 12 cm−1, respectively) for an Fe–OH harmonic oscillator. The measured Fe–O stretching frequency is also in good agreement with the 1.82-Å Fe–O bond reported for CPO-II. A Badger’s rule analysis of this distance provides an Fe–O stretching frequency of νBadger = 563 cm−1. We also present X-band electron nuclear double resonance (ENDOR) data for cryoreduced CPO-II. Cryogenic reduction (77 K) of the EPR-silent Fe(IV)OH center in CPO-II results in an EPR-active Fe(III)OH species with a strongly coupled (13.4 MHz) exchangeable proton. Based on comparisons with alkaline myoglobin, we assign this resonance to the hydroxide proton of cryoreduced CPO-II.

Active Site Threonine Facilitates Proton Transfer During Dioxygen Activation at the Diiron Center of Toluene/o-Xylene Monooxygenase Hydroxylase

Toluene/o-xylene monooxygenase hydroxylase (ToMOH), a diiron-containing enzyme, can activate dioxygen to oxidize aromatic substrates. To elucidate the role of a strictly conserved T201 residue during dioxygen activation of the enzyme, T201S, T201G, T201C, and T201V variants of ToMOH were prepared by site-directed mutagenesis. X-ray crystal structures of all the variants were obtained. Steady-state activity, regiospecificity, and single-turnover yields were also determined for the T201 mutants. Dioxygen activation by the reduced T201 variants was explored by stopped-flow UV-vis and Mössbauer spectroscopy. These studies demonstrate that the dioxygen activation mechanism is preserved in all T201 variants; however, both the formation and decay kinetics of a peroxodiiron(III) intermediate, T201(peroxo), were greatly altered, revealing that T201 is critically involved in dioxygen activation. A comparison of the kinetics of O(2) activation in the T201S, T201C, and T201G variants under various reaction conditions revealed that T201 plays a major role in proton transfer, which is required to generate the peroxodiiron(III) intermediate. A mechanism is postulated for dioxygen activation, and possible structures of oxygenated intermediates are discussed.

Characterization of a peroxodiiron(III) intermediate in the T201S variant of toluene/o-xylene monooxygenase hydroxylase from Pseudomonas sp. OX1.

We report the observation of a novel intermediate in the reaction of a reduced toluene/o-xylene monooxygenase hydroxylase (ToMOH(red)) T201S variant, in the presence of a regulatory protein (ToMOD), with dioxygen. This species is the first oxygenated intermediate with an optical band in any toluene monooxygenase. The UV-vis and Mossbauer spectroscopic properties of the intermediate allow us to assign it as a peroxodiiron(III) species, T201S(peroxo), similar to H(peroxo) in methane monooxygenase. Although T201S generates T201S(peroxo) in addition to optically transparent ToMOH(peroxo), previously observed in wild-type ToMOH, this conservative variant is catalytically active in steady-state catalysis and single-turnover experiments and displays the same regiospecificity for toluene and slightly different regiospecificity for o-xylene oxidation.

Setting an upper limit on the myoglobin iron(IV)hydroxide pK(a): insight into axial ligand tuning in heme protein catalysis.

To provide insight into the iron(IV)hydroxide pKa 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 pKa in myoglobin. Together with the recent determination of an iron(IV)hydroxide pKa ∼ 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.

The Aging-Associated Enzyme CLK-1 Is a Member of the Carboxylate-Bridged Diiron Family of Proteins

The aging-associated enzyme CLK-1 is proposed to be a member of the carboxylate-bridged diiron family of proteins. To evaluate this hypothesis and characterize the protein, we expressed soluble mouse CLK-1 (MCLK1) in Escherichia coli as a heterologous host. Using Mössbauer and EPR spectroscopy, we established that MCLK1 indeed belongs to this protein family. Biochemical analyses of the in vitro activity of MCLK1 with quinone substrates revealed that NADH can serve directly as a reductant for catalytic activation of dioxygen and substrate oxidation by the enzyme, with no requirement for an additional reductase protein component. The direct reaction of NADH with a diiron-containing oxidase enzyme has not previously been encountered for any member of the protein superfamily.