PMC

The electron transfer pathway was obtained after three iterations of the QM/MM e-pathway approach [] with a total of 15 residues (His-304–Arg-311, Leu-323–Ala-325, Leu-373–Gln-375 and Asp-395) included in the quantum region. Each iteration identifies the residue(s) with the highest affinity for the electron, and is shown in a different colour. The mapped route includes Pro-310 and Arg-309, followed by Arg-306, Ile-305 and His-304, as shown by the electron spin distribution.

(A) Total spin density when including Trp-377, Tyr-337 and haem compound I in the quantum region (in the absence of RB19). (B and C) Total spin density when, in addition to RB19 and compound I, the quantum region includes Trp-377 or Tyr-337 respectively. From PDB code 4W7J after 5 ns MD (A) and selected snapshots from two energy minima showing RB19 near Trp-377 and Tyr-337, during PELE [] diffusion in (B and C respectively).

(A) Local minima identified in the PELE [] simulations of RB19 diffusion on the recombinant DyP crystal structure (PDB code 4W7J) showing interaction energy against distance to Tyr-147 (taken as a reference residue). The presence of RB19 in the vicinity of different surface residues and the haem-access channel is indicated. (B) Distances between the closest positions of RB19 (magenta sticks) with respect to haem (1), Tyr-147/Tyr-337 (2), and Trp-377 (3) shown by PELE (A), and between the above residues and the haem cofactor (the distances are measured including hydrogen atoms). RB19 is shown as CPK (Corey–Pauling–Koltun) sticks, Tyr-147/Tyr-337 as magenta sticks and Trp-377 as grey sticks.

(A) General folding constituted by two domains, each of them including two large β-sheets and two or three helices, with the haem cofactor in the upper part of the lower domain [cartoon coloured from the N- to the C-terminus, with the haem shown as CPK (Corey–Pauling–Koltun) sticks]. (B and C) Location of exposed Trp-105, Tyr-147, Trp-207, Tyr-285, Tyr-337 and Trp-377 (as CPK spheres) in two different orientations of the DyP molecule (cartoon with the haem shown as CPK sticks). From PDB code 4W7J.

(A) WT DyP biphasic kinetics enabling calculation of two sets of constants in the 0.2–10 μM (b) and 50–270 μM (a) ranges (inset with Lineweaver–Burk inverse representation for the high, a, and low, b, turnover sites). (B) Simple kinetics yielding a single set of constants (inset, inverse representation) for the W377S variant. (C) Results from Y147S (squares), Y337S (diamonds) and Y147F/Y337F (triangles) variants yielding kinetic curves superimposable with that of WT DyP (circles). (D) Simple kinetics yielding a single set of constants (inset, inverse representation) for the G169L variant. A substrate concentration logarithmic scale is used in the main plots.

(A) Residual activities of 3 μM WT DyP treated with increasing NBS concentrations (in 50 mM acetate, pH 4) for modification of tryptophan residues. (B) Residual activities of 3 μM WT DyP and W377S variant (white and black symbols respectively) treated with increasing TNM concentrations (in 50 mM Tris/HCl, pH 7, with 2.6% ethanol) for modification of tyrosine residues. The residual activities of the WT DyP and the W377S variant in (A) and (B) were monitored for oxidation of 180 μM RB19 (diamonds), 7.5 mM DMP (circles), 15 μM RB5 (squares) and 1.25 mM ABTS (triangles), and referred to activities of untreated WT DyP (taken as 100%).

(A) X-band EPR spectra of WT DyP at pH 3 before (top) and after (bottom) the addition of H₂O₂ (and rapid freezing). Experimental conditions: v=9.39 GHz, 0.2 mW microwave power, 0.4 mT modulation amplitude. (B and C) Narrow scan X-band (v=9.39 GHz) and W-band (v=94.29 GHz) respectively of the radical species. The positions of the three g-tensor components of the tyrosyl contribution are indicated. X-band experimental conditions: v=9.38 GHz, 1 mW microwave power and 0.05 mT modulation amplitude; W-band experimental conditions: v=94.29 GHz, 0.05 mW microwave power and 0.1 mT modulation amplitude. (D) X-band EPR spectrum of the radical intermediate formed in the W377S variant paired with its better simulation (Sim) (see magnetic parameters in Supplementary Table S3). Experimental conditions: v=9.39 GHz, 1 mW microwave power, 0.2 mT modulation amplitude.