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 pK(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 pK(a) in myoglobin. Together with the recent determination of an iron(IV)hydroxide pK(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.

Iron(IV)hydroxide pK(a) and the role of thiolate ligation in C-H bond activation by cytochrome P450.

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 pK(a) (where K(a) 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 pK(a) value for this compound of 11.9. Marcus theory analysis indicates that this elevated pK(a) results in a >10,000-fold reduction in the rate constant for oxidations of the protein framework, making these processes noncompetitive with substrate oxidation.

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.

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.

Reaction of cytochrome P450BM3 and peroxynitrite yields nitrosyl complex.

Peroxynitrite has come into the spotlight in recent years. Its effects on proteins have been implicated in several diseases such as acute lung injury, rheumatoid arthritis, implant rejection, artherosclerosis, Parkinson's disease, and Alzheimer's disease. Peroxynitrite is thought to inactivate a variety of proteins including thiolate-ligated heme proteins such as cytochrome P450 2B1 and PGI2 synthase, through the nitration of tyrosine residues. In previous studies it was reported that thiolate-ligated heme enzymes react with peroxynitrite to form a ferryl intermediate. In an effort to spectroscopically characterize this species in P450BM3, we discovered that the peroxynitrite-generated intermediate is not an FeIVoxo, but rather an iron-nitrosyl [FeNO]6 complex. We present density functional calculations as well as Mössbauer and stopped-flow spectroscopic characterizations of the peroxynitrite-generated intermediate in P450BM3.

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

The electronic properties of an unusually redox-rich iron system, [PhBP(R)3]Fe-Nx (where [PhBP(R)3] 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, [PhBP(iPr)3]Fe[triple bond]N (3a) and [PhBP(CH2Cy)3]Fe[triple bond]N (3b), and the formally diiron(I) bridged-Fe(mu-N2)Fe species, {[PhBP(iPr)3]Fe}2(mu-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 [PhBP(iPr)3]Fe(H)3(PMe3) (5), an S = 3/2 iron(I) phosphine adduct [PhBP(iPr)3]FePMe3 (6), and the S = 2 iron(II) precursors to 3a, [PhBP(iPr)3]Fe-Cl and [PhBP(iPr)3]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 ((18)O and (2)H)-sensitive band at nu(Fe-O) = 565 cm(-1). Preparation of CPO-II in H(2)O using H(2)(18)O(2) results in a red-shift of 22 cm(-1), while preparation of CPO-II in (2)H(2)O using H(2)O(2) 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-A Fe-O bond reported for CPO-II. A Badger's rule analysis of this distance provides an Fe-O stretching frequency of nu(Badger) = 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.

Evidence for basic ferryls in cytochromes P450.

Using a combination of Mössbauer spectroscopy and density functional calculations, we have determined that the ferryl forms of P450(BM3) and P450cam are protonated at physiological pH. Density functional calculations were performed on large active-site models of these enzymes to determine the theoretical Mössbauer parameters for the ferryl and protonated ferryl (Fe(IV)OH) species. These calculations revealed a significant enlargement of the quadrupole splitting parameter upon protonation of the ferryl unit. The calculated quadrupole splittings for the protonated and unprotonated ferryl forms of P450(BM3) are DeltaE(Q) = 2.17 mm/s and DeltaE(Q) = 1.05 mm/s, respectively. For P450cam, they are DeltaE(Q) = 1.84 mm/s and DeltaE(Q) = 0.66 mm/s, respectively. The experimentally determined quadrupole splittings (P450(BM3), DeltaE(Q) = 2.16 mm/s; P450cam, DeltaE(Q) = 2.06 mm/s) are in good agreement with the values calculated for the protonated forms of the enzymes. Our results suggest that basic ferryls are a natural consequence of thiolate-ligated hemes.

Preparation and properties of a monomeric Mn(IV)-oxo complex.

Manganese-oxo complexes have long been investigated because of their proposed roles in biological and chemical catalysis. However, there are few examples of monomeric complexes with terminal oxo ligands, especially those with oxomanganese(IV) units. A oxomanganese(IV) complex has been prepared from [MnIIIH3buea(O)]2- ([H3buea]3-, tris[(N'-tert-butylureaylato)-N-ethylene]aminato), a monomeric MnIII-O complex in which the oxo ligand arises from cleavage of dioxygen. Treating [MnIIIH3buea(O)]2- with [Cp2Fe]BF4 in either DMF at -45 degrees C or DMSO at room temperature produces [MnIVH3buea(O)]-: lambdamax = 635 nm; nu(Mn-16O) = 737 cm-1; nu(Mn-18O) = 709 cm-1; g = 5.15, 2.44, 1.63, D = 3.0 cm-1, E/D = 0.26, aMn = 66 G (A = 190 MHz). These spectroscopic properties support the assignment of a mononuclear MnIV-oxo complex with an S = 3/2 ground state. Density functional theory supports this assignment and the Jahn-Teller distortion around the high-spin MnIV center that would alter the molecular structure of [MnIVH3buea(O)]- from trigonal symmetry (as indicated by the highly rhombic EPR signal). [MnIVH3buea(O)]- is relatively unstable in DMSO, converting to [MnIIIH3buea(OH)]- via a proposed X-H bond cleavage. [MnIVH3buea(O)]- reacts with 1,2-diphenylhydrazine to from azobenzene (95% yield) and [MnIIIH3buea(OH)]-. The MnIV-oxo does not react with triphenyl- or tricyclohexylphosphine. However, O-atom transfer is observed with methyldiphenylphosphine and dimethylphenylphosphine, producing the corresponding phosphine oxides. These results illustrate the diverse reactivity of the MnIV-oxo unit.

Evidence for two ferryl species in chloroperoxidase compound II.

Using a combination of density functional calculations and Mössbauer spectroscopy, we have examined chloroperoxidase compound II (CPO-II). The Mössbauer spectrum of CPO-II suggests the presence of two distinct ferryl species in an approximately 70:30 ratio. Density functional calculations and cryogenic reduction and annealing experiments allow us to assign the major species as an Fe(IV)OH intermediate. The Mössbauer parameters of the minor component are indicative of an authentic iron(IV)oxo species, but we have found the 70:30 ratio to be pH invariant. The unchanging ratio of component concentrations is in agreement with CPO-II's visible absorption spectrum, which shows no change over the enzyme's range of pH stability.

On the status of ferryl protonation.

We examine the issue of ferryl protonation in heme proteins. An analysis of the results obtained from X-ray crystallography, resonance Raman spectroscopy, and extended X-ray absorption spectroscopy (EXAFS) is presented. Fe-O bond distances obtained from all three techniques are compared using Badger's rule. The long Fe-O bond lengths found in the ferryl crystal structures of myoglobin, cytochrome c peroxidase, horseradish peroxidase, and catalase deviate substantially from the values predict by Badger's rule, while the oxo-like distances obtained from EXAFS measurements are in good agreement with the empirical formula. Density functional calculations, which suggest that Mössbauer spectroscopy can be used to determine ferryl protonation states, are presented. Our calculations indicate that the quadrupole splitting (DeltaE(Q)) changes significantly upon ferryl protonation. New resonance Raman data for horse-heart myoglobin compound II (Mb-II, pH 4.5) are also presented. An Fe-O stretching frequency of 790cm(-1) (shifting to 754cm(-1) with (18)O substitution) was obtained. This frequency provides a Badger distance of r(Fe-O)=1.66A. This distance is in agreement with the 1.69A Fe-O bond distance obtained from EXAFS measurements but is significantly shorter than the 1.93A bond found in the crystal structure of Mb-II (pH 5.2). In light of the available evidence, we conclude that the ferryl forms of myoglobin (pKa4), horseradish peroxidase (pKa4), cytochrome c peroxidase (pKa4), and catalase (pKa7) are not basic. They are authentic Fe(IV)oxos with Fe-O bonds on the order of 1.65A.

X-ray absorption spectroscopy of chloroperoxidase compound I: Insight into the reactive intermediate of P450 chemistry.

We report the structural characterization of a thiolate-ligated ferryl radical. Using x-ray absorption spectroscopy, we examined chloroperoxidase (CPO) compound I (CPO-I). Our results indicate that CPO-I is an authentic ferryl species with an Fe-O bond of 1.65 A. Axial-ligand interactions result in a remarkably long 2.48-A Fe-S bond. Analogous forms of cytochrome P450 and CPO have been shown to possess virtually identical coordination environments. Thus, it seems likely that our findings provide a good structural description of the elusive P450-I.