A fluorophore-labeled form of the T4moD, the catalytic effector protein of the toluene 4-monooxygenase complex, was prepared by engineering the N-terminal region to contain a tetraCys motif and treatment with biarsenical fluorescein. Fluorescence anisotropy was used to study the protein-protein interactions among various combinations of the four components of the complex. Binding interactions were detected between T4moD and the hydroxylase component T4moH [K(D) value of 83 nM for interaction with the alphabetagamma protomer] and between T4moD and the Rieske [2Fe-2S] ferredoxin component T4moC (K(D) value of 78 nM). No binding interactions were detected between T4moD and the NADH oxidoreductase component T4moF, but T4moF was able to disrupt binding between T4moC and T4moD. The detected binding interactions suggest an intermediary electron transfer complex between T4moC and T4moD that excludes T4moF. The results indicate that specialization of effector protein function may include specific protein-protein interactions with [2Fe-2S] domains as well as the hydroxylase component.