The assembly of a multisubunit photosynthetic membrane protein complex: a site-specific spin labeling EPR spectroscopic study of the PsaC subunit in photosystem I.
The assembly of the PsaC subunit in the photosystem I (PS I) complex was studied using site-specific 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 C34(C) or the engineered C75(C) residue of wild-type PsaC (PsaC(WT)). Three distinct stages of PsaC assembly were analyzed: unbound PsaC, the P(700)-F(X)/PsaC complex, and the P(700)-F(X)/PsaC/PsaD complex. The changes in the EPR spectral line shape and the rotational correlation time of the spin label when PsaC(WT) binds to the PS I core are consistent with the conformational changes that are expected to occur during the assembly process. The addition of the PsaD subunit to the P(700)-F(X)/PsaC(WT-C34) complex induces further EPR spectral changes, which indicate that the presence of PsaD affects the orientation of the PsaC subunit on the PS I 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 (PsaC(C-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)-F(X)/PsaC(C-term-C34) complex. Because the PsaC(C-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 degrees-rotated, C(2)-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.
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Bharat
B
Jagannathan
Sarah
S
Dekat
John H
JH
Golbeck
K V
KV
Lakshmi
D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D010788:Photosynthesis; D045331:Photosystem I Protein Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013113:Spin Labels; D013379:Substrate Specificity; D046940:Synechococcus / Q000201:enzymology
D008565:Membrane Proteins; D045331:Photosystem I Protein Complex; D021122:Protein Subunits; D013113:Spin Labels; C089909:photosystem I, psaB subunit