Hole Hopping through Cytochrome P450

Abstract

High-potential iron-oxo species are intermediates in the catalytic cycles of oxygenase enzymes. They can cause heme degradation via irreversible oxidation of nearby amino acids. We have proposed that there are protective mechanisms in which hole hopping from oxidized hemes through tryptophan/tyrosine chains generates a surface-exposed amino-acid oxidant that could be rapidly disarmed by reaction with cellular reductants.In investigations of cytochrome P450 BM 3 , we identified Trp96 as a critical residue that could play such a protective role. This Trp is cation-π paired with Arg398 in 81% of mammalian P450s. Here we report on the effect of the Trp/Arg cation-π interaction on Trp96 formal potentials as well as on electronic coupling strengths between Trp96 and the heme both for wild type cytochrome P450 and selected mutants. Mutation of Arg398 to His, which decreases the Trp96 formal potential, increases Trp-heme electronic coupling; but, surprisingly, the rate of phototriggered electron transfer from a Ru-sensitizer (through Trp96) to the P450 BM 3 heme was unaffected by the Arg398His mutation. We conclude that Trp96 has moved away from Arg398, suggesting that the protective mechanism for P450s with this Trp-Arg pair is conformationally gated. photooxidation cycle. Species shown in red represent oxidized intermediates in the reversible cycle. As drawn, the Trp96 Arg398 cation-π interaction would be in close contact as is observed in the crystal structures PDB: 3NPL and 3R1A. The photooxidation cycle begins with a laser pulse that excites the covalently attached Ru(II)photosensitizer, Step 1. This excited species is then quenched oxidatively (Q = Quencher = [Ru(NH 3 ) 6 ]Cl 3 , generating Ru(III)-photosensitizer, Step 2, and triggering the oxidation of Trp, porphyrin, and iron, forming Cpd II, Steps 3-5. The last step involves the reaction of reduced quencher (Q-) with Cpd II and returns the system to its initial state, Step 6. 4

Tryptophan extends the life of cytochrome P450

“…It is noteworthy that in bacterial P450 enzymes, a histidine residue appears more commonly at this location. In eukaryotes, however, tryptophan typically occupies this site ( 38 ). The other two sites in the putative W96|W90|Y334 ETr pathway are far less well conserved (W90, 20%; Y334 65%), implying that multiple hole migration routes to the surface might be available.…”

Tryptophan‐96 in cytochrome P450 BM3 plays a key role in enzyme survival

“…W96 appears to be the gateway residue for the primary Trp/Tyr protection pathway. Sequence alignments suggest that this position is more commonly occupied by a histidine residue in bacterial enzymes, but tryptophan is found at this position in more than 80% of mammalian P450s [16]. High enzyme turnover rates have been associated with increased energy demand and reduced productivity in microbes and plants [17,18].…”