Maraia E. Ener

A serine-substituted P450 catalyzes highly efficient carbene transfer to olefins in vivo

Genetically encoded catalysts for non-natural chemical reactions will open new routes to sustainable production of chemicals. We designed a unique serine-heme ligated cytochrome “P411” that catalyzes efficient and selective carbene transfers from diazoesters to olefins in intact Escherichia coli cells. The mutation C400S in cytochrome P450BM3 gives a signature ferrous-CO Soret peak at 411 nm, abolishes monooxygenation activity, raises the resting state FeIII/II reduction potential, and significantly improves NAD(P)H-driven cyclopropanation activity.

Photooxidation of cytochrome P450-BM3

High-valent iron-oxo species are thought to be intermediates in the catalytic cycles of oxygenases and peroxidases. An attractive route to these iron-oxo intermediates involves laser flash-quench oxidation of ferric hemes, as demonstrated by our work on the ferryl (compound II) and ferryl porphyrin radical cation (compound I) intermediates of horseradish peroxidase. Extension of this work to include cytochrome P450-BM3 (CYP102A1) has required covalent attachment of a RuII photosensitizer to a nonnative cysteine near the heme (), in order to promote electron transfer from the FeIII porphyrin to photogenerated RuIII. The conjugate was structurally characterized by X-ray crystallography (2.4 Å resolution; Ru-Fe distance, 24 Å). Flash-quench oxidation of the ferric-aquo heme produces an FeIV-hydroxide species (compound II) within 2 ms. Difference spectra for three singly oxidized P450-BM3 intermediates were obtained from kinetics modeling of the transient absorption data in combination with generalized singular value decomposition analysis and multiexponential fitting.

Generation of Powerful Tungsten Reductants by Visible Light Excitation

The homoleptic arylisocyanide tungsten complexes, W(CNXy)6 and W(CNIph)6 (Xy = 2,6-dimethylphenyl, Iph = 2,6-diisopropylphenyl), display intense metal to ligand charge transfer (MLCT) absorptions in the visible region (400-550 nm). MLCT emission (λ(max) ≈ 580 nm) in tetrahydrofuran (THF) solution at rt is observed for W(CNXy)6 and W(CNIph)6 with lifetimes of 17 and 73 ns, respectively. Diffusion-controlled energy transfer from electronically excited W(CNIph)6 (*W) to the lowest energy triplet excited state of anthracene (anth) is the dominant quenching pathway in THF solution. Introduction of tetrabutylammonium hexafluorophosphate, [Bu(n)4N][PF6], to the THF solution promotes formation of electron transfer (ET) quenching products, [W(CNIph)6](+) and [anth](•-). ET from *W to benzophenone and cobalticenium also is observed in [Bu(n)4N][PF6]/THF solutions. The estimated reduction potential for the [W(CNIph)6](+)/*W couple is -2.8 V vs Cp2Fe(+/0), establishing W(CNIph)6 as one of the most powerful photoreductants that has been generated with visible light.

Symmetry-Breaking Charge Transfer of Visible Light Absorbing Systems: Zinc Dipyrrins

Zinc dipyrrin complexes with two identical dipyrrin ligands absorb strongly at 450–550 nm and exhibit high fluorescence quantum yields in nonpolar solvents (e.g., 0.16–0.66 in cyclohexane) and weak to nonexistent emission in polar solvents (i.e., <10–3, in acetonitrile). The low quantum efficiencies in polar solvents are attributed to the formation of a nonemissive symmetry-breaking charge transfer (SBCT) state, which is not formed in nonpolar solvents. Analysis using ultrafast spectroscopy shows that in polar solvents the singlet excited state relaxes to the SBCT state in 1.0–5.5 ps and then decays via recombination to the triplet or ground states in 0.9–3.3 ns. In the weakly polar solvent toluene, the equilibrium between a localized excited state and the charge transfer state is established in 11–22 ps.

Fluorescence Quenching of (Dimethylamino)naphthalene Dyes Badan and Prodan by Tryptophan in Cytochromes P450 and Micelles

Fluorescence of 2-(N,N-dimethylamino)-6-propionylnaphthalene dyes Badan and Prodan is quenched by tryptophan in Brij 58 micelles as well as in two cytochrome P450 proteins (CYP102, CYP119) with Badan covalently attached to a cysteine residue. Formation of nonemissive complexes between a dye molecule and tryptophan accounts for about 76% of the fluorescence intensity quenching in micelles, the rest is due to diffusive encounters. In the absence of tryptophan, fluorescence of Badan-labeled cytochromes decays with triexponential kinetics characterized by lifetimes of about 100 ps, 700–800 ps, and 3 ns. Site mutation of a histidine residue in the vicinity of the Badan label by tryptophan results in shortening of all three decay lifetimes. The relative amplitude of the fastest component increases at the expense of the two slower ones. The average quenching rate constants are 4.5 × 108 s–1 (CYP102) and 3.7 × 108 s–1 (CYP119), at 288 K. Cyclic voltammetry of Prodan in MeCN shows a reversible reduction peak at −1.85 V vs NHE that becomes chemically irreversible and shifts positively upon addition of water. A quasireversible reduction at −0.88 V was observed in an aqueous buffer (pH 7.3). The excited-state reduction potential of Prodan (and Badan) is estimated to vary from about +0.6 V (vs NHE) in polar aprotic media (MeCN) to approximately +1.6 V in water. Tryptophan quenching of Badan/Prodan fluorescence in CYPs and Brij 58 micelles is exergonic by ≤0.5 V and involves tryptophan oxidation by excited Badan/Prodan, coupled with a fast reaction between the reduced dye and water. Photoreduction is a new quenching mechanism for 2-(N,N-dimethylamino)-6-propionylnaphthalene dyes that are often used as solvatochromic polarity probes, FRET donors and acceptors, as well as reporters of solvation dynamics.

Hole Hopping through Tryptophan in Cytochrome P450

Electron-transfer kinetics have been measured in four conjugates of cytochrome P450 with surface-bound Ru-photosensitizers. The conjugates are constructed with enzymes from Bacillus megaterium (CYP102A1) and Sulfolobus acidocaldarius (CYP119). A W96 residue lies in the path between Ru and the heme in CYP102A1, whereas H76 is present at the analogous location in CYP119. Two additional conjugates have been prepared with (CYP102A1)W96H and (CYP119)H76W mutant enzymes. Heme oxidation by photochemically generated Ru3+ leads to P450 compound II formation when a tryptophan residue is in the path between Ru and the heme; no heme oxidation is observed when histidine occupies this position. The data indicate that heme oxidation proceeds via two-step tunneling through a tryptophan radical intermediate. In contrast, heme reduction by photochemically generated Ru+ proceeds in a single electron tunneling step with closely similar rate constants for all four conjugates.