Multifrequency ESEEM Spectroscopy of Sulfite Oxidase in Phosphate Buffer: Direct Evidence for Coordinated Phosphate

Abstract

The molybdenum(V) coordination environment of sulfite oxidase has been investigated by multifrequency ESEEM spectroscopy in ∼70 mM phosphate buffer at pH* = 6.5 in both H₂O and D₂O. The FT-ESEEM spectra in H₂O typically consist of three lines. One of these lines is always close to twice the Larmor frequency of the P atom (2ν_P) and is assigned to one or more coordinated phosphates, providing the first direct unambiguous detection of such coordination. Extensive simulations of this phosphate signal at the various operational frequencies indicated that the coordinated phosphate group(s) probably does (do) not adopt a fixed orientation, and as a result, a description of the Mo···P hyperfine interaction required the introduction of a distribution of such orientations, with Mo···P distance(s) of 3.2−3.3 Å. The other two lines in the FT-ESEEM spectra in H₂O, located at ν_H and 2ν_H, were assigned to matrix protons. In D₂O buffer two additional lines, assigned to matrix deuterons, were also seen.