Interaction of the soluble recombinant PsaD subunit of spinach photosystemI with ferredoxin I

Photosystem I of higher plants functions in photosynthesis as a light-drive n oxidoreductase producing reduced ferredoxin. Its peripheral subunit PsaD has been identified as the docking site for ferredoxin I. With the aim of e lucidating the structure-function relationship and the role of this subunit , a recombinant form of the spinach protein was produced by heterologous ex pression in Escherichia coli. The PsaD protein was synthesized in soluble f orm and purified to homogeneity. The interaction of the PsaD subunit with f erredoxin I was investigated using three different approaches: chemical cro ss-linking between the two purified proteins in solution, affinity chromato graphy of the PsaD subunit on a ferredoxin-coupled resin, and titration wit h ferredoxin of the protein fluorescence of the subunit. All these studies indicated that the isolated PsaD in solution has a definite conformation an d maintains the ability to bind ferredoxin I with high affinity and specifi city. The K-d value of the complex of PsaD and ferredoxin is in the nanomol ar range, which is consistent with reported K-m values for ferredoxin I pho toreduction by thylakoid membranes. The ionic strength dependence of the K- d suggests that the protein-protein interaction is at least partially elect rostatic in nature. Nevertheless, none of the glutamate residues of the aci dic cluster of residues 92-94 of ferredoxin I, which have been reported to be involved in the interaction with the subunit, seems to be essential for PsaD binding, as borne out by experiments using ferredoxin I mutants in pos itions 92-94.