Identity of the exchangeable sulfur-containing ligand at the Mo(V) center of R160Q human sulfite oxidase

Abstract

In our previous study of the fatal R160Q mutant of human sulfite oxidase (hSO) at low pH (Astashkin et al. J. Am. Chem. Soc.2008, 130, 8471-8480), a new Mo(V) species, denoted "species 1", was observed at low pH values. Species 1 was ascribed to a six-coordinate Mo(V) center with an exchangeable terminal oxo ligand and an equatorial sulfate group on the basis of pulsed EPR spectroscopy and (33)S and (17)O labeling. Here we report new results for species 1 of R160Q, based on substitution of the sulfur-containing ligand by a phosphate group, pulsed EPR spectroscopy in K(a)- and W-bands, and extensive density functional theory (DFT) calculations applied to large, more realistic molecular models of the enzyme active site. The combined results unambiguously show that species 1 has an equatorial sulfite as the only exchangeable ligand. The two types of (17)O signals that are observed arise from the coordinated and remote oxygen atoms of the sulfite ligand. A typical five-coordinate Mo(V) site is compatible with the observed and calculated EPR parameters.

Figure 1

Active site of SO rendered from the 1.9 Å chicken SO structure, pdb 1SOX. The molybdopterin (MPT), the conserved Mo-bound Cys residue (C185 and C207 in chicken and human SO, respectively), the axial and equatorial oxo ligands, and Mo are shown as ball-and-stick (blue-green = Mo, red = O, yellow = S, gray = C, orange = P, and purple = N). The protein is displayed as ribbons and a cross-section to reveal the surfaces of the substrate and cofactor pockets and their relative orientations.

Figure 2

K_a-band ESE-detected field sweep spectra of R160Q hSO (primary ESE). (a) Solid trace, the spectrum of the original sample at pH = 6.4, which contains contributions from Species 1 (long-dashed trace) and Species 2 (short-dashed trace). (b) Solid trace, the spectrum obtained after adding phosphate buffer, where the final concentration of phosphate was ~200 mM. The short- and long-dashed traces show the contributions from Species 2 and 1P (formed from Species 1). The spectrum of Species 1P (long-dashed) is the difference between the solid and short-dashed traces. Experimental conditions: mw frequency = 30.068 GHz; mw pulses, 2×12 ns; time interval between the mw pulses, τ = 200 ns; temperature = 21 K. The arrow shows the EPR position at which the ESEEM spectra of Figures 3 and 4 were obtained (B_o = 1074 mT).

Figure 3

K_a-band two-pulse ESEEM spectra (cosine FT) of Species 1 (upper trace) and Species 1P (lower trace) obtained at the EPR position indicated by the arrow in Figure 2. Experimental conditions: mw frequency = 30.068 GHz; B_o = 1074 mT; mw pulses, 2×12 ns; temperature = 21 K. For clarity, the modulation amplitudes to the right of the break in the x-axis have been magnified by a factor of 3. The proton matrix line is marked by the arrow labeled “¹H” (at ~46 MHz, which corresponds to the ¹H Larmor frequency, ν_H). Likewise, the arrow labeled “³¹P” indicates the position of the sum combination line of ³¹P (ν_σ = ~37.5 MHz), which is close to double the Larmor frequency of ³¹P at this magnetic field, 2ν_P ≈ 37.1 MHz.

Figure 4

K_a-band HYSCORE spectrum of Species 1P obtained at the EPR position indicated by the arrow in Figure 2. Experimental conditions: mw frequency = 30.068 GHz; B_o = 1074 mT; mw pulses, 12, 12, 22, and 12 ns; temperature = 21 K. The spectrum represents the sum of the spectra recorded at the time intervals (τ) between the first two mw pulses for 150, 180, and 210 ns. The arrows labeled “³¹P” point at the ³¹P fundamental lines, and the arrows labeled “¹⁷O” indicate the lines of ¹⁷O from H-bonded water.

Figure 5

ED-NMR spectra of Species 1 measured at different magnetic field positions (indicated on the left side of each trace). The largest hyperfine coupling (observed at 3449 mT) is labeled by “*”. The spectral features of the weakly coupled ¹⁷O (observable on the same trace and labeled by “O”) are centered about the ¹⁷O Larmor frequency (ν_O ~ 20 MHz) and are separated by ~4.5 MHz. The field positions of g_x, g_y, and g_z that correspond to the labeled traces are shown in Figure S6. Experimental conditions: preparation (probing) mw pulse = 10 μs; the observation two-pulse sequence consisted of 100 ns (π/2) and 200 ns (π) pulses separated by a time interval (τ) of 400 ns. The time interval between the preparation and observation pulses was 10 μs; observation mw frequency = 94.897 GHz; temperature = 21 K.

Figure 6

W-band HYSCORE spectra of Species 1 obtained at the g_y (a) and g_x (b) EPR spectrum positions. The spectra represent the sums of the spectra that were obtained at time intervals (τ) between the first two mw pulses of 137 and 175 ns. The correlation lines of coordinated and remote ¹⁷O are indicated. Experimental conditions: mw frequency = 94.897 GHz; B_o = 3441.7 mT (a) and 3472.9 mT (b); mw pulses = 12.5, 12.5, 25, and 12.5 ns; temperature = 21 K.

Figure 7

Sum combination line region of the cosine FT spectra of the two-pulse W- and integrated four-pulse K_a-band ESEEM^, (traces 1 and 2, respectively) obtained at g_x. The quadrupole splittings, Δν_Q, are indicated for each trace. Experimental conditions for trace 1: mw frequency = 94.897 GHz; B_o = 3472.9 mT; mw pulses, 2×25 ns; temperature= 21 K. Experimental conditions for trace 2: mw frequency = 29.523 GHz; B_o = 1081 mT; mw pulses, 4×15 ns; temperature = 21 K. A frequency of zero is defined as double the ¹⁷O Zeeman frequency at the given magnetic fields.

Figure 8

W-band two-pulse ESEEM spectrum of Species 1 recorded at g_y. The ¹⁷O Larmor (ν_O) and double Larmor (2ν_O) frequencies, and the hfi constant (A) of the coordinated ¹⁷O are labeled. Experimental conditions: mw pulse durations = 12.5 ns (π/2) and 25 ns (π); mw frequency = 94.897 GHz; B_o = 3441.7 mT; temperature = 21 K.

Figure 9

Stereo view (cross-eye) representations of the energy-minimized sulfate- and sulfite-bound SO models (upper and lower, respectively). The magnetic resonance parameters calculated for these models are provided in Table 1, and the atom coordinates are provided in the Supporting Information. blue-green = Mo, red = O, yellow = S, gray = C, and blue = N.

Scheme 1

Preparations of the various R160Q hSO species and schematic representations of each of their 5-coordinate Mo centers. The straight lines in the chemical structures denote the coordinated sulfur atoms from the conserved cysteine and the MPT (Figure 1).