Intramolecular electron transfer (IET) between the molybdenumandheme centers of vertebrate sulfite oxidase (SO) is proposed to bea key step in the catalytic cycle of the enzyme. However, the X-raycrystallographic distance between these centers, RMoFe= 32.3 Å, appears to be too long for the rapidIET rates observed in liquid solution. The Mo and heme domains arelinked by a flexible tether, and it has been proposed that dynamicinterdomain motion brings the two metal centers closer together andthereby facilitates rapid IET. To date, there have been no directdistance measurements for SO in solution that would support or contradictthis model. In this work, pulsed electron–electron double resonance(ELDOR) and relaxation induced dipolar modulation enhancement (RIDME)techniques were used to obtain information about RMoFein the Mo(V)Fe(III) state of wild type recombinanthuman SO in frozen glassy solution. Surprisingly, the data obtainedsuggest a fixed structure with RMoFe=32 Å, similar to that determined by X-ray crystallography forchicken SO, although the orientation of the RMoFeradius-vector with respect to the heme center wasfound to be somewhat different. The implications of these findingsfor the flexible tether model are discussed. [ABSTRACT FROM AUTHOR]