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 and 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_BM3, 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; however, surprisingly, the rate of phototriggered electron transfer from a Ru-sensitizer (through Trp96) to the P450_BM3 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.

FIGURE. 1:

RuP450_BM3 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(NH₃)₆]Cl₃, 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.

FIGURE. 2:

Conformational changes between the open and closed conformations of P450_2b4 from 3R1A (left) to 1PO5 (right). Region with the most change is colored maroon, heme (white and blue), Fe (orange), Arginine (yellow and blue), Tryptophan (magenta and blue). Notably, portions of the C helix containing Trp120 (Trp96 is P450_BM3 numbering) move, dramatically increasing the Trp-Arg distance. Helices with significant conformational change are labelled.

FIGURE. 3:

Geometry of: 1PO5 (blue), 2BDM (magenta), 3R1B (orange), 3R1A (purple), 3G5N (pink), 3G93 (white), and 1DT6 (green).

FIGURE. 4:

A schematic view of the electron transfer step, for which we calculate the electronic coupling elements. An electron is transferred from the porphyrin ring of the heme to the tryptophan cation, Trp^•+.

FIGURE. 5:

Relative orientations of Trp residues (3NPL structure): (A) single Trp; (B) Trp/Arg cluster; (C) Trp/Arg/heme-substituent (acid group) cluster; and (D) Trp/Arg/heme cluster.

FIGURE. 6:

(Top) TA kinetics traces for wild type and R398H RuP450_BM3 light red (R398H, 390 nm), dark red (wt, 390 nm), light green (R398H, 420 nm), dark green (wt, 420 nm), light blue (R398H, 440 nm), dark blue (wt, 440 nm). (Bottom) wild type RuP450_BM3 luminescence decay, (left); wild type RuP450_BM3 R398H luminescence decay, (right).

FIGURE. 7:

Absolute reduction potential (ΔE) (eV) of each of the four cluster sizes, shown in Figure 5, for three different geometries of P450 both the wild type (in greens) and the His mutation (in pinks). Calculations performed with the PCM solvent model.

FIGURE. 8:

Absolute reduction potential (ΔE) (eV), of cluster B (light green), and cluster C (dark green), as shown in Figure 5. Various P450 geometries shown in relation to the Arg-Trp distance (Å). Fit of a function of the form $\frac{a}{r}+b$ to the Cluster B absolute reduction potentials (pink).