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

The molybdenum(V) coordination environment of sulfite oxidase has been investigated by multifrequency ESEEM spectroscopy in approximately 70 mM phosphate buffer at pH = 6.5 in both H(2)O and D(2)O. The FT-ESEEM spectra in H(2)O typically consist of three lines. One of these lines is always close to twice the Larmor frequency of the P atom (2nu(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 H(2)O, located at nu(H) and 2nu(H), were assigned to matrix protons. In D(2)O buffer two additional lines, assigned to matrix deuterons, were also seen.