Alistair J. Fielding

Identification and structural basis of the reaction catalyzed by CYP121, an essential cytochrome P450 in Mycobacterium tuberculosis.

The gene encoding the cytochrome P450 CYP121 is essential for Mycobacterium tuberculosis. However, the CYP121 catalytic activity remains unknown. Here, we show that the cyclodipeptide cyclo(l-Tyr-l-Tyr) (cYY) binds to CYP121, and is efficiently converted into a single major product in a CYP121 activity assay containing spinach ferredoxin and ferredoxin reductase. NMR spectroscopy analysis of the reaction product shows that CYP121 catalyzes the formation of an intramolecular C-C bond between 2 tyrosyl carbon atoms of cYY resulting in a novel chemical entity. The X-ray structure of cYY-bound CYP121, solved at high resolution (1.4 Å), reveals one cYY molecule with full occupancy in the large active site cavity. One cYY tyrosyl approaches the heme and establishes a specific H-bonding network with Ser-237, Gln-385, Arg-386, and 3 water molecules, including the sixth iron ligand. These observations are consistent with low temperature EPR spectra of cYY-bound CYP121 showing a change in the heme environment with the persistence of the sixth heme iron ligand. As the carbon atoms involved in the final C-C coupling are located 5.4 Å apart according to the CYP121-cYY complex crystal structure, we propose that C-C coupling is concomitant with substrate tyrosyl movements. This study provides insight into the catalytic activity, mechanism, and biological function of CYP121. Also, it provides clues for rational design of putative CYP121 substrate-based antimycobacterial agents.

Neutron cryo-crystallography captures the protonation state of ferryl heme in a peroxidase

Peroxidase proton placement Heme enzymes catalyze a variety of biochemical oxidations through the activation of oxygen by iron. Casadei et al. used neutron crystallography to elucidate the mechanism of cytochrome c peroxidase (see the perspective by Groves and Boaz). In the highly reactive intermediate state termed compound I, the iron(IV) oxo, or ferryl, fragment was not protonated, whereas a nearby histidine residue was protonated. The sensitivity of neutron scattering to proton locations revealed these protonation states, where more common techniques, such as x-ray diffraction, have yielded more ambiguous results. Science, this issue p. 193; see also p. 142 The sensitivity of neutron scattering to proton locations clarifies the acid/base chemistry of a biochemical oxidation. [Also see Perspective by Groves and Boaz] Heme enzymes activate oxygen through formation of transient iron-oxo (ferryl) intermediates of the heme iron. A long-standing question has been the nature of the iron-oxygen bond and, in particular, the protonation state. We present neutron structures of the ferric derivative of cytochrome c peroxidase and its ferryl intermediate; these allow direct visualization of protonation states. We demonstrate that the ferryl heme is an Fe(IV)=O species and is not protonated. Comparison of the structures shows that the distal histidine becomes protonated on formation of the ferryl intermediate, which has implications for the understanding of O–O bond cleavage in heme enzymes. The structures highlight the advantages of neutron cryo-crystallography in probing reaction mechanisms and visualizing protonation states in enzyme intermediates.

Impact of molecular size on electron spin relaxation rates of nitroxyl radicals in glassy solvents between 100 and 300 K

Electron spin lattice relaxation rates were measured for 12 nitroxyls with molecular weights between 144 and 438, and for galvinoxyl, 1,3-bisdiphenylene-2-phenylallyl (BDPA), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) in glassy sucrose octaacetate. Relaxation rates for polar nitroxyls also were measured in glassy sorbitol. Dependence on T where V eff is effective molecular volume and γ is a material-specific parameter, was used to compare processes. Values of V eff were determined based on molecular libration in glassy sucrose octaacetate (γ = 3.5), tumbling in viscous decalin at 233 K (γ = 4.7), or tumbling in heavy mineral oil (γ = 6.0). For nitroxyl relaxation there is a master curve: log(1/T 1) vs log( T) (γ = 0.89). The similarity of the values of γ for the Raman process and for the additional process that contributes at higher temperatures, and the absence of frequency dependence between X- and Q-band, support assignment of this additional process as a local mode. For these radicals the contributions from the local mode and the Raman process are correlated and follow trends in spin–orbit coupling. The temperature dependence of spin echo dephasing in sucrose octaacetate is dominated by rotation of methyl groups and by a motional process analogous to the Raman process.

Multifrequency EPR of four triarylmethyl radicals

Continuous-wave spectra at W-band of four triarylmethyl (trityl) radicals at 100 K in 1∶1 water-glycerol exhibit rhombic electron paramagnetic resonance spectra. The rigid-lattice line widths at W-band are only 3 to 5 times larger than at X-band or S-band, and fluid-solution line widths are much narrower than those for rigid lattice, which indicates that unresolved anisotropic nuclear hyperfine couplings make significant contributions to the rigid-lattice line widths. Spin-flip lines are observed in glassy-solution spectra at X-band and S-band, but not at W-band or 250 MHz. At 100 KTm is dominated by spin diffusion of solvent protons and is independent of microwave frequency. Between about 130 and 170 K, 1/Tm for trityl-CH3 is enhanced by rotation of the methyl groups at a rate comparable to inequivalences in the hyperfine interaction. Motional averaging of anisotropic interactions enhances spin echo dephasing between about 200 and 300 K. The temperature dependence of 1/T1 is similar for the four radicals and is consistent with assignment of the Raman process and a local mode as the dominant relaxation processes. The similarity inT1 values at W-band and X-band supports this assignment.

New Developments in Spin Labels for Pulsed Dipolar EPR

Spin labelling is a chemical technique that enables the integration of a molecule containing an unpaired electron into another framework for study. Given the need to understand the structure, dynamics, and conformational changes of biomacromolecules, spin labelling provides a relatively non-intrusive technique and has certain advantages over X-ray crystallography; which requires high quality crystals. The technique relies on the design of binding probes that target a functional group, for example, the thiol group of a cysteine residue within a protein. The unpaired electron is typically supplied through a nitroxide radical and sterically shielded to preserve stability. Pulsed electron paramagnetic resonance (EPR) techniques allow small magnetic couplings to be measured (e.g., <50 MHz) providing information on single label probes or the dipolar coupling between multiple labels. In particular, distances between spin labels pairs can be derived which has led to many protein/enzymes and nucleotides being studied. Here, we summarise recent examples of spin labels used for pulse EPR that serve to illustrate the contribution of chemistry to advancing discoveries in this field.

Intramolecular electron transfer versus substrate oxidation in lactoperoxidase: investigation of radical intermediates by stopped-flow absorption spectrophotometry and (9-285 GHz) electron paramagnetic resonance spectroscopy.

We have combined the information obtained from rapid-scan electronic absorption spectrophotometry and multifrequency (9-295 GHz) electron paramagnetic resonance (EPR) spectroscopy to unequivocally determine the electronic nature of the intermediates in milk lactoperoxidase as a function of pH and to monitor their reactivity with organic substrates selected by their different accessibilities to the heme site. The aim was to address the question of the putative catalytic role of the protein-based radicals. This experimental approach allowed us to discriminate between the protein-based radical intermediates and [Fe(IV)=O] species, as well as to directly detect the oxidation products by EPR. The advantageous resolution of the g anisotropy of the Tyr (*) EPR spectrum at high fields showed that the tyrosine of the [Fe(IV)=O Tyr (*)] intermediate has an electropositive and pH-dependent microenvironment [g(x) value of 2.0077(0) at pH >or= 8.0 and 2.0066(2) at 4.0 <or= pH <or= 7.5] possibly related to the radical stability and function. Two types of organic molecules (small aromatic vs bulkier substrates) allowed us to distinguish different mechanisms for substrate oxidation. [Fe(IV)=O Por (*+)] is the oxidizing species of benzohydroxamic acid, o-dianisidine, and o-anisidine via a heme-edge reaction and of mitoxantrone via a long-range electron transfer (favored at pH 8) not involving the tyrosyl radical, the formation of which competed with the substrate oxidation at pH 5. In contrast, the very efficient reaction with ABTS at pH 5 is consistent with [Fe(IV)=O Tyr (*)] being the oxidizing species. Accordingly, the identification of the ABTS binding site by X-ray crystallography may be a valuable tool in rational drug design.

Enzymatic single-molecule kinetic isotope effects

Ensemble-based measurements of kinetic isotope effects (KIEs) have advanced physical understanding of enzyme-catalyzed reactions, but controversies remain. KIEs are used as reporters of rate-limiting H-transfer steps, quantum mechanical tunnelling, dynamics and multiple reactive states. Single molecule (SM) enzymatic KIEs could provide new information on the physical basis of enzyme catalysis. Here, single pair fluorescence energy transfer (spFRET) was used to measure SM enzymatic KIEs on the H-transfer catalyzed by the enzyme pentaerythritol tetranitrate reductase. We evaluated a range of methods for extracting the SM KIE from single molecule spFRET time traces. The SM KIE enabled separation of contributions from nonenzymatic protein and fluorophore processes and H-transfer reactions. Our work demonstrates SM KIE analysis as a new method for deconvolving reaction chemistry from intrinsic dynamics.

Multifrequency electron paramagnetic resonance characterization of PpoA, a CYP450 fusion protein that catalyzes fatty acid dioxygenation.

PpoA is a fungal dioxygenase that produces hydroxylated fatty acids involved in the regulation of the life cycle and secondary metabolism of Aspergillus nidulans . It was recently proposed that this novel enzyme employs two different heme domains to catalyze two separate reactions: within a heme peroxidase domain, linoleic acid is oxidized to (8R)-hydroperoxyoctadecadienoic acid [(8R)-HPODE]; in the second reaction step (8R)-HPODE is isomerized within a P450 heme thiolate domain to 5,8-dihydroxyoctadecadienoic acid. In the present study, pulsed EPR methods were applied to find spectroscopic evidence for the reaction mechanism, thought to involve paramagnetic intermediates. We observe EPR resonances of two distinct heme centers with g-values typical for Fe(III) S = (5)/(2) high-spin (HS) and Fe(III) S = (1)/(2) low-spin (LS) hemes. (14)N ENDOR spectroscopy on the S = (5)/(2) signal reveals resonances consistent with an axial histidine ligation. Reaction of PpoA with the substrate leads to the formation of an amino acid radical on the early millisecond time scale concomitant to a substantial reduction of the S = (5)/(2) heme signal. High-frequency EPR (95- and 180-GHz) unambiguously identifies the new radical as a tyrosyl, based on g-values and hyperfine couplings from spectral simulations. The radical displays enhanced T(1)-spin-lattice relaxation due to the proximity of the heme centers. Further, EPR distance measurements revealed that the radical is distributed among the monomeric subunits of the tetrameric enzyme at a distance of approximately 5 nm. The identification of three active paramagnetic centers involved in the reaction of PpoA supports the previously proposed reaction mechanism based on radical chemistry.

Catalytic Amine Oxidation under Ambient Aerobic Conditions: Mimicry of Monoamine Oxidase B†

The flavoenzyme monoamine oxidase (MAO) regulates mammalian behavioral patterns by modulating neurotransmitters such as adrenaline and serotonin. The mechanistic basis which underpins this enzyme is far from agreed upon. Reported herein is that the combination of a synthetic flavin and alloxan generates a catalyst system which facilitates biomimetic amine oxidation. Mechanistic and electron paramagnetic (EPR) spectroscopic data supports the conclusion that the reaction proceeds through a radical manifold. This data provides the first example of a biorelevant synthetic model for monoamine oxidase B activity.

A Rapid Freeze‐Quench Setup for Multi‐Frequency EPR Spectroscopy of Enzymatic Reactions

Electron paramagnetic resonance (EPR) spectroscopy in combination with the rapid freeze-quench (RFQ) technique is a well-established method to trap and characterize intermediates in chemical or enzymatic reactions at the millisecond or even shorter time scales. The method is particularly powerful for mechanistic studies of enzymatic reactions when combined with high-frequency EPR (ν≥90 GHz), which permits the identification of substrate or protein radical intermediates by their electronic g values. In this work, we describe a new custom-designed micro-mix rapid freeze-quench apparatus, for which reagent volumes for biological samples as small as 20 μL are required. The apparatus was implemented with homemade sample collectors appropriate for 9, 34, and 94 GHz EPR capillaries (4, 2, and 0.87 mm outer diameter, respectively) and the performance was evaluated. We demonstrate the application potential of the RFQ apparatus by following the enzymatic reaction of PpoA, a fungal dioxygenase producing hydro(pero)xylated fatty acids. The larger spectral resolution at 94 GHz allows the discernment of structural changes in the EPR spectra, which are not detectable in the same samples at the standard 9 GHz frequency.