A multifrequency electron paramagnetic resonance (EPR) approach was used to probe the dynamic structure of the bacterial photosynthetic reaction center (RC) protein. We have demonstrated that the cysteine specific nitroxide spin label MTSL can be covalently bound to a surface cysteine residue of Rhodobacter sphaeroides RC protein. We suggest that the MTSL nitroxide is bound to an accessible cysteine residue, H156, which is located on the surface of the protein on the H-subunit. Analysis of the multifrequency EPR spectra of the spin-labeled RC proteins suggests the restricted character of the protein dynamics. These dynamics can be described as fast libration in a cone with a correlation time faster than 10(-9) s. Several dynamically nonequivalent sites were observed in the EPR spectra, which may reflect distinct conformational substates of local protein structure. This work provides a foundation for future studies with the goal of correlating protein motions with photosynthetic charge-transfer reactions.