MODIFIED LIGANDS TO F-A AND F-B IN PHOTOSYSTEM-I - PROPOSED CHEMICAL RESCUE OF A [4FE-4S] CLUSTER WITH AN EXTERNAL THIOLATE IN ALANINE, GLYCINE, AND SERINE MUTANTS OF PSAC

The F-B and F-A electron accepters in Photosystem I (PS I) are [4Fe-4S ] clusters ligated by cysteines provided by PsaC. In a previous study (Mehari, T., Qiao, F., Scott, M. P., Nellis, D., Zhao, J., Bryant, D., and Golbeck, J. H. (1995) J. Biol. Chem. 270, 28108-28117), we showed that when cysteines 14 and 51 were replaced with serine or alanine, t he free proteins contained a S = 1/2, [4Fe-4S] cluster at the unmodifi ed site and a mixed population of S = 1/2, [3Fe-4S] and S = 3/2, [4Fe- 4S] clusters at the modified site. We show here that these mutant PsaC proteins can be rebound to P700-F-X cores, resulting in fully functio nal PS I complexes. The low temperature EPR spectra of the C14X(PsaC). PS I complexes (where X = S, A, or G) show the photoreduction of a wi ld-type F-A cluster and a modified F-B cluster, the latter with g valu es of 2.115, 1.899, and 1.852 and linewidths of 110, 70, and 85 MHz. S ince neither alanine nor glycine contains a suitable side group, an ex ternal thiolate provided by beta-mercaptoethanol has likely been recru ited to supply the requisite ligand to the [4Fe-4S] cluster. The EPR s pectrum of the C51S(PsaC). PS I complex differs from that of the C51A( PsaC). PS I or C51G(PSaC). PS I complexes by the presence of an additi onal set of resonances, which may be derived from the serine oxygen-li gated cluster. In all other mutant PS I complexes, a wild-type spin-co upled interaction spectrum appears when FA and F, are simultaneously r educed. Single turnover flash studies indicate similar to 50% efficien t electron transfer to F-A/F-B in the C14SP(Sac). PS I, C51S(PsaC). PS I, C14G(PSaC). PS I, and CB14S(PsaC). PS I mutants and less than 40% in the C14A(PsaC). PS I and C51A(PsaC). PS I mutants, compared with si milar to 76% in the PS I core reconstructed with wild-type PsaC. These data are consistent with the measurements of the rates of cytochrome c(6)-NADP(+) reductase activity, indicating lower rates in the alanine mutants. It is proposed that the chemical rescue of a [4Fe-4S] cluste r with a recruited external thiolate at the modified site allows the m utant PsaC proteins to rebind to PS I and to function in forward elect ron transfer.