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A requirement for an active proton delivery network supports a compound I-mediated C-C bond cleavage in CYP51 catalysis.

ABSTRACT: CYP51 enzymes (sterol 14α-demethylases) are cytochromes P450 that catalyze multistep reactions. The CYP51 reaction occurs in all biological kingdoms and is essential in sterol biosynthesis. It removes the 14α-methyl group from cyclized sterol precursors by first forming an alcohol, then an aldehyde, and finally eliminating formic acid with the introduction of a Δ14-15 double bond in the sterol core. The first two steps are typical hydroxylations, mediated by an electrophilic compound I mechanism. The third step, C-C bond cleavage, has been proposed to involve either compound I (i.e. FeO₃ ⁺) or, alternatively, a proton transfer-independent nucleophilic ferric peroxo anion (compound 0, i.e. Fe₃ ⁺O₂ ^-). Here, using comparative crystallographic and biochemical analyses of WT human CYP51 (CYP51A1) and its D231A/H314A mutant, whose proton delivery network is destroyed (as evidenced in a 1.98-Å X-ray structure in complex with lanosterol), we demonstrate that deformylation of the 14α-carboxaldehyde intermediate requires an active proton relay network to drive the catalysis. These results indicate a unified, compound I-based mechanism for all three steps of the CYP51 reaction, as previously established for CYP11A1 and CYP19A1. We anticipate that our approach can be applied to mechanistic studies of other P450s that catalyze multistep reactions, such as C-C bond cleavage.

SUBMITTER: Hargrove TY

PROVIDER: S-EPMC7380200 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Concerted heavy-atom bond cleavage and proton and electron transfers illustrated by proton-assisted reductive cleavage of an O-O bond.

Project description:Electron transfer may be concerted with proton transfer. It may also be concerted with the cleavage of a bond between heavy atoms. All three events may also be concerted. A model is presented to analyze the kinetics of these all-concerted reactions for homogeneous or electrochemical reduction or oxidation processes. It allows the estimation of the kinetic advantage that derives from the increase of the bond-breaking driving force resulting from the concerted proton transfer. Application of the model to the electrochemical reductive cleavage of the O-O bond of an organic peroxide in the presence of a proximal acid group illustrates the applicability of the model and provides an example demonstrating that electron transfer, heavy-atom bond breaking, and proton transfer may be all concerted. Such analyses are expected to be useful for the invention, analysis, and optimization of reactions involved in contemporary energy challenges as well as for the comprehension of major biochemical processes, a number of which involve electron and proton transfer together with cleavage of bonds between heavy atoms.

| S-EPMC3102404 | biostudies-literature

Transition State Asymmetry in C-H Bond Cleavage by Proton-Coupled Electron Transfer.

Project description:The selective transformation of C-H bonds is a longstanding challenge in modern chemistry. A recent report details C-H oxidation via multiple-site concerted proton-electron transfer (MS-CPET), where the proton and electron in the C-H bond are transferred to separate sites. Reactivity at a specific C-H bond was achieved by appropriate positioning of an internal benzoate base. Here, we extend that report to reactions of a series of molecules with differently substituted fluorenyl-benzoates and varying outer-sphere oxidants. These results probe the fundamental rate versus driving force relationships in this MS-CPET reaction at carbon by separately modulating the driving force for the proton and electron transfer components. The rate constants depend strongly on the pKa of the internal base, but depend much less on the nature of the outer-sphere oxidant. These observations suggest that the transition states for these reactions are imbalanced. Density functional theory (DFT) was used to generate an internal reaction coordinate, which qualitatively reproduced the experimental observation of a transition state imbalance. Thus, in this system, homolytic C-H bond cleavage involves concerted but asynchronous transfer of the H+ and e-. The nature of this transfer has implications for synthetic methodology and biological systems.

| S-EPMC7241443 | biostudies-literature

Entropic origin of cobalt-carbon bond cleavage catalysis in adenosylcobalamin-dependent ethanolamine ammonia-lyase.

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| S-EPMC3839591 | biostudies-literature

Isotope-Labeling Studies Support the Electrophilic Compound I Iron Active Species, FeO(3+), for the Carbon-Carbon Bond Cleavage Reaction of the Cholesterol Side-Chain Cleavage Enzyme, Cytochrome P450 11A1.

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| S-EPMC5289895 | biostudies-literature

Structural characterization of Class 2 OLD family nucleases supports a two-metal catalysis mechanism for cleavage.

Project description:Overcoming lysogenization defect (OLD) proteins constitute a family of uncharacterized nucleases present in bacteria, archaea, and some viruses. These enzymes contain an N-terminal ATPase domain and a C-terminal Toprim domain common amongst replication, recombination, and repair proteins. The in vivo activities of OLD proteins remain poorly understood and no definitive structural information exists. Here we identify and define two classes of OLD proteins based on differences in gene neighborhood and amino acid sequence conservation and present the crystal structures of the catalytic C-terminal regions from the Burkholderia pseudomallei and Xanthamonas campestris p.v. campestris Class 2 OLD proteins at 2.24 Å and 1.86 Å resolution respectively. The structures reveal a two-domain architecture containing a Toprim domain with altered architecture and a unique helical domain. Conserved side chains contributed by both domains coordinate two bound magnesium ions in the active site of B. pseudomallei OLD in a geometry that supports a two-metal catalysis mechanism for cleavage. The spatial organization of these domains additionally suggests a novel mode of DNA binding that is distinct from other Toprim containing proteins. Together, these findings define the fundamental structural properties of the OLD family catalytic core and the underlying mechanism controlling nuclease activity.

| S-EPMC6755086 | biostudies-literature

Theoretical Study of C-H Bond Cleavage via Concerted Proton-Coupled Electron Transfer in Fluorenyl-Benzoates.

Project description:Developing new strategies to activate and cleave C-H bonds is important for a broad range of applications. Recently a new approach for C-H bond activation using multi-site concerted proton-coupled electron transfer (PCET) involving intermolecular electron transfer to an oxidant coupled to intramolecular proton transfer was reported. For a series of oxidants reacting with 2-(9 H-fluoren-9-yl)benzoate, experimental studies revealed an atypical Brønsted ?, defined as the slope of the logarithm of the PCET rate constant versus the logarithm of the equilibrium constant or the scaled driving force. Herein this reaction is modeled with a vibronically nonadiabatic PCET theory. Hydrogen tunneling, thermal sampling of the proton donor-acceptor mode, solute and solvent reorganization, and contributions from excited vibronic states are found to play important roles. The calculations qualitatively reproduce the experimental observation of a Brønsted ? significantly less than 0.5 and explain this shallow slope in terms of exoergic processes between pairs of electron-proton vibronic states. These fundamental mechanistic insights may guide the design of more effective strategies for C-H bond activation and cleavage.

| S-EPMC6402776 | biostudies-literature

Use of a novel fluorinated organosulfur compound to isolate bacteria capable of carbon-sulfur bond cleavage.

Project description:The vacuum residue fraction of heavy crudes contributes to the viscosity of these oils. Specific microbial cleavage of C-S bonds in alkylsulfide bridges that form linkages in this fraction may result in dramatic viscosity reduction. To date, no bacterial strains have been shown conclusively to cleave C-S bonds within alkyl chains. Screening for microbes that can perform this activity was greatly facilitated by the use of a newly synthesized compound, bis-(3-pentafluorophenylpropyl)-sulfide (PFPS), as a novel sulfur source. The terminal pentafluorinated aromatic rings of PFPS preclude growth of aromatic ring-degrading bacteria but allow for selective enrichment of strains capable of cleaving C-S bonds. A unique bacterial strain, Rhodococcus sp. strain JVH1, that used PFPS as a sole sulfur source was isolated from an oil-contaminated environment. Gas chromatography-mass spectrometry analysis revealed that JVH1 oxidized PFPS to a sulfoxide and then a sulfone prior to cleaving the C-S bond to form an alcohol and, presumably, a sulfinate from which sulfur could be extracted for growth. Four known dibenzothiophene-desulfurizing strains, including Rhodococcus sp. strain IGTS8, were all unable to cleave the C-S bond in PFPS but could oxidize PFPS to the sulfone via the sulfoxide. Conversely, JVH1 was unable to oxidize dibenzothiophene but was able to use a variety of alkyl sulfides, in addition to PFPS, as sole sulfur sources. Overall, PFPS is an excellent tool for isolating bacteria capable of cleaving subterminal C-S bonds within alkyl chains. The type of desulfurization displayed by JVH1 differs significantly from previously described reaction results.

| S-EPMC368330 | biostudies-literature

Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site.

Project description:Hydrogen bond networks are key elements of protein structure and function but have been challenging to study within the complex protein environment. We have carried out in-depth interrogations of the proton transfer equilibrium within a hydrogen bond network formed to bound phenols in the active site of ketosteroid isomerase. We systematically varied the proton affinity of the phenol using differing electron-withdrawing substituents and incorporated site-specific NMR and IR probes to quantitatively map the proton and charge rearrangements within the network that accompany incremental increases in phenol proton affinity. The observed ionization changes were accurately described by a simple equilibrium proton transfer model that strongly suggests the intrinsic proton affinity of one of the Tyr residues in the network, Tyr16, does not remain constant but rather systematically increases due to weakening of the phenol-Tyr16 anion hydrogen bond with increasing phenol proton affinity. Using vibrational Stark spectroscopy, we quantified the electrostatic field changes within the surrounding active site that accompany these rearrangements within the network. We were able to model these changes accurately using continuum electrostatic calculations, suggesting a high degree of conformational restriction within the protein matrix. Our study affords direct insight into the physical and energetic properties of a hydrogen bond network within a protein interior and provides an example of a highly controlled system with minimal conformational rearrangements in which the observed physical changes can be accurately modeled by theoretical calculations.

| S-EPMC3710806 | biostudies-literature

Strategies for Enhancing the Rate Constant of C-H Bond Cleavage by Concerted Proton-Coupled Electron Transfer.

Project description:Recently selective C-H bond cleavage under mild conditions with weak oxidants was reported for fluorenyl-benzoates. This mechanism is based on multi-site concerted proton-coupled electron transfer (PCET) involving intermolecular electron transfer to an outer-sphere oxidant coupled to intramolecular proton transfer to a well-positioned proton acceptor. The electron transfer driving force depends predominantly on the oxidant, and the proton transfer driving force depends mainly on the basicity of the carboxylate, which is influenced by the substituent on the benzoate fragment. Experiments showed that the rate constants are much more sensitive to the carboxylate basicity than to the redox potential of the oxidant. Herein a vibronically nonadiabatic PCET theory is used to explain how changing the driving force for the electron and proton transfer components of the reaction through varying the oxidant and the substituent, respectively, impacts the PCET rate constant. In addition to increasing the driving force for proton transfer, enhancing the basicity of the carboxylate also decreases the equilibrium proton donor-acceptor distance, thereby facilitating the sampling of shorter proton donor-acceptor distances. This additional effect arising from the strong dependence of proton transfer on the proton donor-acceptor distance provides an explanation for the greater sensitivity of the rate constant to the carboxylate basicity than to the redox potential of the oxidant. These fundamental insights have broad implications for developing new strategies to activate C-H bonds, specifically by designing systems with shorter equilibrium proton donor-acceptor distances.

| S-EPMC6927470 | biostudies-literature

Facile C-H bond cleavage via a proton-coupled electron transfer involving a C-H...Cu(II) interaction.

Project description:The present study provides mechanistic details of a mild aromatic C-H activation effected by a copper(II) center ligated in a triazamacrocylic ligand, affording equimolar amounts of a Cu(III)-aryl species and Cu(I) species as reaction products. At low temperatures the Cu(II) complex 1 forms a three-center, three-electron C-H...Cu(II) interaction, identified by pulse electron paramagnetic resonance spectroscopy and supported by density functional theory calculations. C-H bond cleavage is coupled with copper oxidation, as a Cu(III)-aryl product 2 is formed. This reaction proceeds to completion at 273 K within minutes through either a copper disproportionation reaction or, alternatively, even faster with 1 equiv of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), quantitatively yielding 2. Kinetic studies of both reactions strongly implicate a rate-limiting proton-coupled electron transfer as the key C-H activation step, a mechanism that does not conform to the C-H activation mechanism in a Ni(II) analogue or to any previously proposed C-H activation mechanisms.

| S-EPMC2937838 | biostudies-literature

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