Michael T. Green

Cytochrome P450 compound I: capture, characterization, and C-H bond activation kinetics.

The Power Behind P450 Drugs, toxins and a range of metabolic substrates are detoxified in the liver by family of iron-containing enzymes called cytochrome P450. The iron component transfers oxygen to compounds that are often highly resistant to chemical reaction, but we know very little about the mechanism of this vital detoxification process. Rittle and Green (p. 933; see the Perspective by Sligar) have managed to capture the P450 reaction intermediate by freezing a solution of the enzyme as it reacts with an oxidant. Spectroscopic techniques and kinetic studies then revealed an iron(IV)oxo intermediate that passes its oxygen along to the substrate with remarkable speed. The iron-bound oxygen intermediate responsible for many challenging biochemical oxidations has been thoroughly characterized. Cytochrome P450 enzymes are responsible for the phase I metabolism of approximately 75% of known pharmaceuticals. P450s perform this and other important biological functions through the controlled activation of C-H bonds. Here, we report the spectroscopic and kinetic characterization of the long-sought principal intermediate involved in this process, P450 compound I (P450-I), which we prepared in approximately 75% yield by reacting ferric CYP119 with m-chloroperbenzoic acid. The Mössbauer spectrum of CYP119-I is similar to that of chloroperoxidase compound I, although its electron paramagnetic resonance spectrum reflects an increase in |J|/D, the ratio of the exchange coupling to the zero-field splitting. CYP119-I hydroxylates the unactivated C-H bonds of lauric acid [D(C-H) ~ 100 kilocalories per mole], with an apparent second-order rate constant of kapp = 1.1 × 107 per molar per second at 4°C. Direct measurements put a lower limit of k ≥ 210 per second on the rate constant for bound substrate oxidation, whereas analyses involving kinetic isotope effects predict a value in excess of 1400 per second.

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.

CH bond activation in heme proteins: the role of thiolate ligation in cytochrome P450

The ability of cytochrome P450 to functionalize unactivated hydrocarbons at physiological temperature and pressure has attracted considerable interest from the chemical community. One of the more intriguing aspects of cytochrome P450 is the enzymes use of a thiolate-ligated heme to perform demanding two-electron oxidations. This coordination is unusual, given that thiolate ligation can significantly decrease a heme reduction potential. In an effort to understand Natures use of a donating thiolate in cytochrome P450, we have undertaken a systematic study of the high-valent forms of thiolate ligated heme proteins. Our investigations have revealed that the ferryl forms of these enzymes are basic. The basic ferryls afforded by thiolate ligation bias cytochrome P450 toward H-atom abstraction, creating an oxidant that cleaves CH bonds while avoiding unwanted oxidations of the protein superstructure. Recent synthetic work supports this hypothesis.

Reactive Intermediates in Cytochrome P450 Catalysis

Recently, we reported the spectroscopic and kinetic characterizations of cytochrome P450 compound I in CYP119A1, effectively closing the catalytic cycle of cytochrome P450-mediated hydroxylations. In this minireview, we focus on the developments that made this breakthrough possible. We examine the importance of enzyme purification in the quest for reactive intermediates and report the preparation of compound I in a second P450 (P450ST). In an effort to bring clarity to the field, we also examine the validity of controversial reports claiming the production of P450 compound I through the use of peroxynitrite and laser flash photolysis.

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.

Cytochrome P450: the active oxidant and its spectrum.

This Forum focuses on the nature of the elusive oxidizing intermediate in P450 catalysis. The identity of this species has reemerged as a topic of contentious debate. It was recently reported that laser flash photolysis (LFP) can be used to generate P450 compound I (P450-I) quantitatively. Kinetic analyses of the reaction of the LFP-generated intermediate with substrates have been suggested to indicate that compound I is not the active oxidant in P450 catalysis. We evaluate these claims via an analysis of the UV/visible spectrum of the LFP-generated intermediate. The techniques of singular value decomposition and target testing are used to obtain the spectrum of P450-I in a model-independent manner from stopped-flow data of the reaction of P450 with m-chloroperbenzoic acid. It is shown that the LFP-generated spectrum bears no similarity to the P450-I spectrum. One may conclude that the LFP-generated intermediate is not P450-I.

Structural analysis of the Mn(IV)/Fe(III) cofactor of Chlamydia trachomatis ribonucleotide reductase by extended X-ray absorption fine structure spectroscopy and density functional theory calculations.

The class Ic ribonucleotide reductase from Chlamydia trachomatis ( Ct) uses a stable Mn(IV)/Fe(III) cofactor to initiate nucleotide reduction by a free-radical mechanism. Extended X-ray absorption fine structure (EXAFS) spectroscopy and density functional theory (DFT) calculations are used to postulate a structure for this cofactor. Fe and Mn K-edge EXAFS data yield an intermetallic distance of approximately 2.92 A. The Mn data also suggest the presence of a short 1.74 A Mn-O bond. These metrics are compared to the results of DFT calculations on 12 cofactor models derived from the crystal structure of the inactive Fe 2(III/III) form of the protein. Models are differentiated by the protonation states of their bridging and terminal OH X ligands as well as the location of the Mn(IV) ion (site 1 or 2). The models that agree best with experimental observation feature a mu-1,3-carboxylate bridge (E120), terminal solvent (H 2O/OH) to site 1, one mu-O bridge, and one mu-OH bridge. The site-placement of the metal ions cannot be discerned from the available data.

Rational Tuning of the Thiolate Donor in Model Complexes of Superoxide Reductase: Direct Evidence for a Trans Influence in FeIII-OOR Complexes

Iron peroxide species have been identified as important intermediates in a number of nonheme iron as well as heme-containing enzymes, yet there are only a few examples of such species either synthetic or biological that have been well characterized. We describe the synthesis and structural characterization of a new series of five-coordinate (N4S(thiolate))Fe(II) complexes that react with tert-butyl hydroperoxide ((t)BuOOH) or cumenyl hydroperoxide (CmOOH) to give metastable alkylperoxo-iron(III) species (N4S(thiolate)Fe(III)-OOR) at low temperature. These complexes were designed specifically to mimic the nonheme iron active site of superoxide reductase, which contains a five-coordinate iron(II) center bound by one Cys and four His residues in the active form of the protein. The structures of the Fe(II) complexes are analyzed by X-ray crystallography, and their electrochemical properties are assessed by cyclic voltammetry. For the Fe(III)-OOR species, low-temperature UV-vis spectra reveal intense peaks between 500-550 nm that are typical of peroxide to iron(III) ligand-to-metal charge-transfer (LMCT) transitions, and EPR spectroscopy shows that these alkylperoxo species are all low-spin iron(III) complexes. Identification of the vibrational modes of the Fe(III)-OOR unit comes from resonance Raman (RR) spectroscopy, which shows nu(Fe-O) modes between 600-635 cm(-1) and nu(O-O) bands near 800 cm(-1). These Fe-O stretching frequencies are significantly lower than those found in other low-spin Fe(III)-OOR complexes. Trends in the data conclusively show that this weakening of the Fe-O bond arises from a trans influence of the thiolate donor, and density functional theory (DFT) calculations support these findings. These results suggest a role for the cysteine ligand in SOR, and are discussed in light of the recent assessments of the function of the cysteine ligand in this enzyme.

Cryoreduction of the NO-Adduct of Taurine:α-Ketoglutarate Dioxygenase (TauD) Yields an Elusive {FeNO}8 Species

The Fe(II)- and alpha-ketoglutarate (alphaKG)-dependent enzymes are a functionally and mechanistically diverse group of mononuclear nonheme-iron enzymes that activate dioxygen to couple the decarboxylation of alphaKG, which yields succinate and CO(2), to the oxidation of an aliphatic C-H bond of their substrates. Their mechanisms have been studied in detail by a combination of kinetic, spectroscopic, and computational methods. Two reaction intermediates have been trapped and characterized for several members of this enzyme family. The first intermediate is the C-H-cleaving Fe(IV)-oxo complex, which exhibits a large deuterium kinetic isotope effect on its decay. The second intermediate is a Fe(II):product complex. Reaction intermediates proposed to occur before the Fe(IV)-oxo intermediate do not accumulate and therefore cannot be characterized experimentally. One of these intermediates is the initial O(2) adduct, which is a {FeO(2)}(8) species in the notation introduced by Enemark and Feltham. Here, we report spectroscopic and computational studies on the stable NO-adduct of taurine:alphaKG dioxygenase (TauD), termed TauD-{FeNO}(7), and its one-electron reduced form, TauD-{FeNO}(8). The latter is isoelectronic with the proposed O(2) adduct and was generated by low-temperature gamma-irradiation of TauD-{FeNO}(7). To our knowledge, TauD-{FeNO}(8) is the first paramagnetic {FeNO}(8) complex. The detailed analysis of experimental and computational results shows that TauD-{FeNO}(8) has a triplet ground state. This has mechanistic implications that are discussed in this Article. Annealing of the triplet {FeNO}(8) species presumably leads to an equally elusive {FeHNO}(8) complex with a quintet ground state.