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Hole Hopping through Tryptophan in Cytochrome P450.

ABSTRACT: 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.

SUBMITTER: Ener ME

PROVIDER: S-EPMC5518273 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Hole Hopping through Tryptophan in Cytochrome P450.

Ener Maraia E ME Gray Harry B HB Winkler Jay R JR

Biochemistry 20170709 28

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 photochemica ...[more]

PMID: 28689401

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Project description:We have investigated photoinduced hole hopping in a Pseudomonas aeruginosa azurin mutant Re126WWCuI, where two adjacent tryptophan residues (W124 and W122) are inserted between the CuI center and a Re photosensitizer coordinated to a H126 imidazole (Re = ReI(H126)(CO)3(dmp)+, dmp = 4,7-dimethyl-1,10-phenanthroline). Optical excitation of this mutant in aqueous media (≤40 μM) triggers 70 ns electron transport over 23 Å, yielding a long-lived (120 μs) ReI(H126)(CO)3(dmp•-)WWCuII product. The Re126FWCuI mutant (F124, W122) is not redox-active under these conditions. Upon increasing the concentration to 0.2-2 mM, {Re126WWCuI}2 and {Re126FWCuI}2 are formed with the dmp ligand of the Re photooxidant of one molecule in close contact (3.8 Å) with the W122' indole on the neighboring chain. In addition, {Re126WWCuI}2 contains an interfacial tryptophan quadruplex of four indoles (3.3-3.7 Å apart). In both mutants, dimerization opens an intermolecular W122' → //*Re ET channel (// denotes the protein interface, *Re is the optically excited sensitizer). Excited-state relaxation and ET occur together in two steps (time constants of ∼600 ps and ∼8 ns) that lead to a charge-separated state containing a Re(H126)(CO)3(dmp•-)//(W122•+)' unit; then (CuI)' is oxidized intramolecularly (60-90 ns) by (W122•+)', forming ReI(H126)(CO)3(dmp•-)WWCuI//(CuII)'. The photocycle is closed by ∼1.6 μs ReI(H126)(CO)3(dmp•-) → //(CuII)' back ET that occurs over 12 Å, in contrast to the 23 Å, 120 μs step in Re126WWCuI. Importantly, dimerization makes Re126FWCuI photoreactive and, as in the case of {Re126WWCuI}2, channels the photoproduced "hole" to the molecule that was not initially photoexcited, thereby shortening the lifetime of ReI(H126)(CO)3(dmp•-)//CuII. Although two adjacent W124 and W122 indoles dramatically enhance CuI → *Re intramolecular multistep ET, the tryptophan quadruplex in {Re126WWCuI}2 does not accelerate intermolecular electron transport; instead, it acts as a hole storage and crossover unit between inter- and intramolecular ET pathways. Irradiation of {Re126WWCuII}2 or {Re126FWCuII}2 also triggers intermolecular W122' → //*Re ET, and the Re(H126)(CO)3(dmp•-)//(W122•+)' charge-separated state decays to the ground state by ∼50 ns ReI(H126)(CO)3(dmp•-)+ → //(W122•+)' intermolecular charge recombination. Our findings shed light on the factors that control interfacial hole/electron hopping in protein complexes and on the role of aromatic amino acids in accelerating long-range electron transport.

Dataset Information

Hole Hopping through Tryptophan in Cytochrome P450.

Publications

Hole Hopping through Tryptophan in Cytochrome P450.

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