A comparative analysis of the spin state distribution of in vitro and in vivo mutants of PsaC. A biochemical argument for the sequence of electron transfer in Photosystem I as F(X) → F(A) → F(B) → ferredoxin/flavodoxin

Abstract

The EPR properties of in vivo and in vitro C14X–PS I and C51X–PS I (X = D, S, A or G) mutants of PsaC are compared in an attempt to extract information about electron transfer not contained in any one of these studies in isolation. This analysis indicates that 1) sulfur from an external 'rescue thiolate' is preferred over oxygen from an aspartate or serine replacement amino acid as a ligand to the FA and FB iron-sulfur clusters; 2) the inherent spectroscopic symmetry in the FA and FB clusters of unbound PsaC is lost when PsaC is docked to its site on the PsaA/PsaB heterodimer; 3) the bound 'rescue thiolate' ligand in the modified site of the FA cluster, but not the FB, cluster is displaced when PsaC is docked to its site on the PsaA/PsaB heterodimer; 4) the free energy of binding PsaC to the PsaA/PsaB heterodimer drives the otherwise-unfavorable ligand replacement in the FA site. These and other findings argue that the substitute ligands support a [4Fe–4S] cluster at the modified site, but the cluster is in either a ground spin state of S ≥ 3/2 or S = 1/2 depending on the chemical identity of the ligand, on whether PsaC is unbound or bound, and on the reduction state of the cluster in the unmodified site. By a comparative analysis of the spin state distribution of the in vivo and in vitro C14X–PS I and C51X–PS I (X = D, S, A or G) mutants, and with knowledge from the X-ray crystal structure that PsaC is bound asymmetrically to the PS I reaction center, an independent case is made that PsaC is oriented so that the FA cluster is proximal to FX and the FB cluster is distal to FX. These results are compared and contrasted with the results of in vivo mutagenesis studies of PsaC in Anabaena variabilis ATCC 29413 and in Chlamydomonas reinhardtii. In all cases, the primary data can be interpreted to support the sequence of electron transfer as FX → FA → FB → ferredoxin.