The assembly of the PsaC subunit in the photosystem I (PS I) complex was studied using sitespecific spin labeling electron paramagnetic resonance (EPR) spectroscopic techniques. The binding was monitored, from the perspective of a reporter spin label attached to either the native C34c or the engineered C75c residue of wild-type PsaC (PsaCWT)- Three distinct stages of PsaC assembly were analyzed: unbound PsaC, the Pvoo-Fx/PsaC complex, and the P700-Fx/PsaC/PsaD complex. The changes in the EPR spectral line shape and. the rotational correlation time of the spin label when PsaCWT binds to the PSI core are consistent with the conformational changes that are expected to occur during the assembly process. The addition of the PsaD subunit to the P700-Fx/PsaCWT-c34 complex induces further EPR spectral, changes, which indicate that the presence of PsaD affects the orientation of the PsaC subunit on the PSI core. The binding of several PsaC variants, each, lacking one or more key binding contacts with, the PsaA/PsaB heterodimer, was monitored using a reporter spin label at C34 c. Our results indicate that the absence of the PsaC-PsaA/PsaB binding contacts causes PsaC to bind in an altered configuration on the PS I core. In particular, the removal of the entire C-terminus (PsaCc-term causes PsaC to dock in a significantly different orientation when compared to the wild-type protein, as indicated by the EPR spectrum of the P 700-Fx/PsaCc-term-C34 complex. Because the PsaCC-term variant retains only the symmetric network of PsaC-PsaA/PsaB ionic contacts, the altered. EPR spectrum could, in principle, reflect a fraction of reaction centers that contain PsaC bound in the 180°-rotated, C2-symmetry-related configuration. The results of this study are used to provide a comprehensive, stepwise mechanism for the binding of PsaC on the PS I core.
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