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 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 autooxidation of tyrosine and tryptophan residues in the enzyme’s framework. Here we report 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.
Aliphatic halogenases activate O2, cleave α-ketoglutarate (αKG) to CO2 and succinate, and form haloferryl [X-Fe(IV)=O; X = Cl, Br] complexes that cleave aliphatic C-H bonds to install halogens during the biosynthesis of natural products by non-ribosomal peptide synthetases (NRPSs). For the related αKG-dependent dioxygenases, it has been shown that reaction of the Fe(II) cofactor with O2 to form the C-H-cleaving ferryl complex is “triggered” by binding of the target substrate. In this study, we have tested for and defined structural determinants of substrate triggering (ST) in the halogenase, SyrB2, from the syringomycin E biosynthetic NRPS of Pseudomonas syringae B301D. As for other halogen ases, the substrate of SyrB2 is complex, consisting of l-Thr tethered via thioester linkage to a covalently bound phosphopantetheine (PPant) cofactor of a carrier protein, SyrB1. Without an appended amino acid, SyrB1 does not trigger formation of the chloroferryl intermediate state in SyrB2, even in the presence of free l-Thr or its analogues, but SyrB1 charged either by l-Thr or by any of several non-native amino acids does trigger the reaction by as much as 8,000-fold (for l-Thr-S-SyrB1). Triggering efficacy is sensitive to the structures of both the amino acid and the carrier protein, being diminished by 5–20-fold when the native l-Thr is replaced by another amino acid and by ∼ 40-fold when SyrB1 is replaced by a heterologous carrier protein, CytC2. The directing effect of the carrier protein and consequent tolerance for profound modifications to the target amino acid allow the chloroferryl state to be formed in the presence of substrates that perturb the ratio of its two putative coordination isomers, lack the target C-H bond (l-Ala-S-SyrB1), or contain a C-H bond of enhanced strength (l-cyclopropylglycyl-S-SyrB1). For the latter two cases, the SyrB2 chloroferryl state so formed exhibits unprecedented stability (t1/2 = 30 – 110 min at 0 °C), can be trapped in high concentration and purity by manual freezing without a cryo-solvent, and represents an ideal target for structural characterization. As initial steps toward this goal, extended x-ray absorption fine structure (EXAFS) spectroscopy has been used to determine the Fe-O and Fe-Cl distances and density functional theory (DFT) calculations have been used to confirm that the measured distances are consistent with the anticipated structure of the intermediate.
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 (P450 ST ). 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.A significant amount of research in the field of bioinorganic chemistry is focused on discerning the intimate details of enzyme catalysis. Critical to these efforts is the preparation of reactive intermediates that form the stations of the catalytic cycle of an enzyme. The study of these transient species is driven by the hope that insights gleaned from their electronic, structural, and kinetic characterizations will guide the design of next-generation catalysts or point the way to inhibitors that could serve as drugs for a variety of maladies. Although a thorough characterization of all the intermediates in a catalytic cycle is required for a detailed dissection of the catalytic mechanism, there are certain species that have special significance and, as such, are considered high-value targets for characterization. These species are generally the highly reactive intermediates that are ultimately responsible for the most important, difficult, or chemically interesting transformation in the catalytic mechanism.Recently, we reported the capture and characterization of one of the most highly sought intermediates in biological chemistry, P450 compound I (P450-I) 4 (1). This iron(IV)-oxo (or ferryl) radical species (7 in Fig. 1) had long been thought to be the principal intermediate in cytochrome P450 catalysis, but due to its highly reactive nature, it had eluded definitive characterization for over 40 years. The existence of P450-I was postulated based on the observation of a "shunt pathway" (Fig. 1) allowing the oxidation of substrates through the use of oxygen donors such as hydrogen peroxide and meta-chloroperbenzoic acid. These oxidants were known to generate high-valent iron-oxo species in heme peroxidases (2, 3), suggesting that a similar intermediate might be involved in P450 catalysis. However, 4 decades worth of searching for the elusive P450-I had led to questions about not only its competence as a hydroxylating agent but also its role in P450 catalysis (4 -6).Our investigations confirmed the existence and the reactive nature of the intermediate. P450-I is capable of hydroxylating unactivated C-H bonds with the remarkable rate constant of 1 ϫ 10 7 M Ϫ1 s Ϫ1 (1). Kinetic isotope effects support a mechanism in which P450-I abstracts hydrogen from substrate, forming an iron(IV)-hydroxide complex that rapidly recombines with substrate to yield hydroxylated product (7-9 in Fig. 1...
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 ~2.92 Å. The Mn data also suggest the presence of a short 1.74 Å 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 Fe2(III/III) form of the protein. Models are differentiated by the protonation states of their bridging and terminal OHX 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 µ-1,3-carboxylate bridge (E120), terminal solvent (H2O/OH) to site 1, one µ-O bridge, and one µ-OH bridge. The site-placement of the metal ions cannot be discerned from the available data.
Biocatalysis by nitrogenase, particularly the reduction of N 2 and CO by this enzyme, has tremendous significance in environmentand energy-related areas. Elucidation of the detailed mechanism of nitrogenase has been hampered by the inability to trap substrates or intermediates in a well-defined state. Here, we report the capture of substrate CO on the resting-state vanadium-nitrogenase in a catalytically competent conformation. The close resemblance of this active CO-bound conformation to the recently described structure of CO-inhibited molybdenum-nitrogenase points to the mechanistic relevance of sulfur displacement to the activation of iron sites in the cofactor for CO binding. Moreover, the ability of vanadium-nitrogenase to bind substrate in the resting-state uncouples substrate binding from subsequent turnover, providing a platform for generation of defined intermediate(s) of both CO and N 2 reduction.nitrogenase | vanadium | carbon monoxide | turnover | substrate binding N itrogenases are complex metalloenzymes that catalyze the reduction of a variety of substrates under ambient conditions (1-3). Among them, two reactions bear significant relevance to environment-and energy-related areas: (i) the reduction of dinitrogen (N 2 ), a key element of nitrogen cycle in our biosphere, to the bio-accessible form of ammonia (NH 3 ); and (ii) the reduction of carbon monoxide (CO), a waste product from car and factory exhausts, to useful hydrocarbon products. The molybdenum (Mo)-and vanadium (V)-nitrogenases are two homologous members of this enzyme family. Both enzymes consist of a reductase component (nifH-or vnfH-encoded Fe protein) and a catalytic component (nifDK-encoded MoFe protein or vnfDGK-encoded VFe protein). Substrate turnover by both nitrogenases involves the formation of a functional complex between the two component proteins (1-3), which enables adenosine triphosphate (ATP)-dependent, interprotein transfer of electrons from the reductase component to the cofactor site of the catalytic component for the subsequent reduction of substrates.Designated the M and V cluster, respectively, the cofactors of Mo-and V-nitrogenases closely resemble each other in geometry (3-7). Previous Fe K-edge X-ray absorption spectroscopy (XAS)/ extended X-ray absorption fine structure (EXAFS) analysis revealed that the two cofactors had nearly indistinguishable metalsulfur core structures, each comprising MFe 3 S 3 (M = Mo or V) and Fe 4 S 3 subclusters bridged by three μ 2 -coordinated, belt-sulfur (S) atoms ( Fig. 1 A and B) (3, 6, 8). Recently, we performed a K-valence X-ray emission spectroscopy (XES) study of both protein-bound and solvent-extracted V clusters (Fig. 1C), which identified a carbide (C 4-)-specific XES feature of the V cluster that was observed earlier in the case of the M cluster (9). Thus, the two cofactors not only share an overall homology in structure, but also have the same inner strengths that originate from the μ 6 -coordinated interstitial carbide. Surprisingly, despite the significant homology bet...
Cytochrome P450 (P450) and chloroperoxidase (CPO) are thiolate ligated heme proteins that catalyze the activation of carbon hydrogen bonds. The principal intermediate in these reactions is a ferryl radical species called compound I. P450 compound I (P450-I) is significantly more reactive than CPO-I, which only cleaves activated C-H bonds. To provide insight into the differing reactivities of these intermediates, we examined CPO-I and P450-I with variable temperature Mössbauer and X-ray absorption spectroscopies. These measurements indicate that the Fe-S bond is significantly shorter in P450-I than in CPO-I. This difference in Fe-S bond lengths can be understood in terms of variations in hydrogen bonding patterns within the “cys-pocket” (a portion of the proximal helix that encircles the thiolate ligand). Weaker hydrogen bonding in P450-I results in a shorter Fe-S bond, which enables greater electron donation from the axial-thiolate ligand. This observation may in part explain P450's greater propensity for C-H bond activation.
To understand how hierarchically structured functional materials operate, analytical tools are needed that can reveal small structural and chemical details in large sample volumes. Often, a single method alone is not sufficient to get a complete picture of processes happening at multiple length scales. Here we present a correlative approach combining three-dimensional X-ray imaging techniques at different length scales for the analysis of metal poisoning of an individual catalyst particle. The correlative nature of the data allowed establishing a macro-pore network model that interprets metal accumulations as a resistance to mass transport and can, by tuning the effect of metal deposition, simulate the response of the network to a virtual ageing of the catalyst particle. The developed approach is generally applicable and provides an unprecedented view on dynamic changes in a material's pore space, which is an essential factor in the rational design of functional porous materials.
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.
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