Determination of the electron self-exchange rates of blue copper proteins by super-WEFT NMR spectroscopy

An NMR approach for determining the electron self-exchange (ESE) rate const ants in blue copper proteins is presented. The approach uses the paramagnet ic relaxation enhancement of resonances in 1D H-1 super-WEFT spectra of par tly oxidized (paramagnetic) proteins. These spectra allow a more precise de termination of the relevant paramagnetic linebroadenings than conventional 1D H-1 spectra and, thus, permit a more detailed investigation of the appli cability of the linebroadenings for determining the electron exchange rates . The approach was used to estimate the ESE rate constant of plastocyanin f rom Anabaena variabilis. It was found that, although the rate constant can be determined accurately from a series of resonances, precise but erroneous constants are obtained from the resonances of the copper-bound residues, u nless a narrow splitting of these resonances caused by the presence of two conformations is taken into account. As demonstrated here, this complicatio n can be overcome by a correct analysis of the paramagnetic broadening of t he combined double signals. Because of the high resolution and specific sen sitivity of the approach it should be generally applicable to estimate elec tron transfer rates, k, if the paramagnetic relaxation enhancement R-2p of the resonances can be determined, and the conditions k much less thanR(2p) or Delta omega (p)>>k>>R-2p are fulfilled, Delta omega (p) being the freque ncy separation between corresponding diamagnetic and paramagnetic sites.