Selective incorporation of 5-hydroxytryptophan blocks long range electron transfer in oxalate decarboxylase

Abstract

Oxalate decarboxylase from Bacillus subtilis is a binuclear Mn-dependent acid stress response enzyme that converts the mono-anion of oxalic acid into formate and carbon dioxide in a redox neutral unimolecular disproportionation reaction. A π-stacked tryptophan dimer, W96 and W274, at the interface between two monomer subunits facilitates long-range electron transfer between the two Mn ions and plays an important role in the catalytic mechanism. Substitution of W96 with the unnatural amino acid 5-hydroxytryptophan leads to a persistent EPR signal which can be traced back to the neutral radical of 5-hydroxytryptophan with its hydroxyl proton removed. 5-Hydroxytryptophan acts as a hole sink preventing the formation of Mn(III) at the N-terminal active site and strongly suppresses enzymatic activity. The lower boundary of the standard reduction potential for the active site Mn(II)/Mn(III) couple can therefore be estimated as 740 mV against the normal hydrogen electrode at pH 4, the pH of maximum catalytic efficiency. Our results support the catalytic importance of long-range electron transfer in oxalate decarboxylase while at the same time highlighting the utility of unnatural amino acid incorporation and specifically the use of 5-hydroxytryptophan as an energetic sink for hole hopping to probe electron transfer in redox proteins.

Keywords: 5-hydroxytryptophan; density functional theory; electron paramagnetic resonance; genetic code expansion; long range electron transfer; oxalate decarboxylase.

SCHEME 1

Decarboxylase activity of OxDC: Two Mn ions are cofactors in a single OxDC subunit. The resting state of the active site is Mn(II) while Mn(III) catalyzes the initial oxidation of the substrate (Twahir et al., 2016). The pH is raised by consumption of the acidic proton of the substrate and formation of a covalent C—H bond in the product, formate

FIGURE 1

(a) Trimer half of the hexameric quaternary assembly. In the crystal structure two of these trimers are stacked face‐to‐face to form the hexamer in the unit cell. The bicupin monomers are shown in green, cyan, and magenta colors. The Mn ions are shown as purple (N‐terminal) and brown (C‐terminal) spheres. (b) Interface between the green and magenta sub‐unit in (a) showing the 4‐TRP box with the π‐stacked dimer, W96 and W274, as well as monomers W171 and W348. Their colors (in part b) correspond to the respective subunit colors (in part a). Distances are measured from the center of the indole rings and are given in units of Å. Both figures used the low‐pH crystal structure of OxDC, 5VG3, and were drawn using PyMol

FIGURE 2

CW EPR spectra of the 5‐HTP substituted mutant enzyme at pH 4.5 in X‐band before (black spectrum) and after reaction with substrate oxalate. The red, blue, and green spectra are scans after the reaction was stopped by freezing in liquid nitrogen after a cumulative reaction time of 1.25 min, 6.0 min, and 33 min, respectively. Enzyme concentration was 0.3 mM and initial substrate concentration was set at 50 mM. The EPR parameters were 9.6506 GHz frequency, 10 G modulation amplitude (part a) and 4 G modulation amplitude (part b). The modulation frequency was 100 kHz and the microwave power 0.6325 mW. The temperature was set at 6 K. (a) Wide field sweep. (b) Narrow sweep around the g = 2 region

FIGURE 3

Comparison between the EPR spectra of the 5‐HTP substituted mutant enzyme with fully protonated 5‐HTP and partially deuterated 5‐HTP in the g ≈ 2 region. EPR parameters are the same for both spectra as given for Figure 2b

FIGURE 4

DFT model of the OxDC active site with the labels of the amino acids as defined in the 5VG3 crystal structure from B. subtilis. Hydrogen atoms bonded to carbon atoms are omitted for clarity

FIGURE 5

Total spin density distribution plot for the OxDC model of the 5HTP/W274 neutral radical with the hydroxide proton of the 5‐HTP removed