Modified ligands to F sub(A) and F sub(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 sub(B) and F sub(A) electron acceptors in Photosystem I (PS I) are [4Fe-4S] clusters ligated by cysteines provided by PsaC. In a previous study, we showed that when cysteines 14 and 51 were replaced with serine or alanine, the free proteins contained a S = 1/2, [4Fe-4S] cluster at the unmodified 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 sub(x) cores, resulting in fully functional PS I complexes. The low temperature EPR spectra of the C14X sub(PsaC)*PS I complexes (where X = S, A, or G) show the photoreduction of a wild-type F sub(A) cluster and a modified F sub(B) cluster, the latter with g values of 2.115, 1.899, and 1.852 and linewidths of 110, 70, and 85 MHz. Since neither alanine nor glycine contains a suitable side group, an external thiolate provided by beta -mercaptoethanol has likely been recruited to supply the requisite ligand to the [4Fe-4S] cluster. The EPR spectrum of the C51S sub(PsaC)*PS I complex differs from that of the C51A sub(PsaC)*PS I or C51G sub(PsaC)*PS I complexes by the presence of an additional set of resonances, which may be derived from the serine oxygen-ligated cluster. In all other mutant PS I complexes, a wild-type spin-coupled interaction spectrum appears when F sub(A) and F sub(B) are simultaneously reduced. Single turnover flash studies indicate similar to 50% efficient electron transfer to F sub(A) /F sub(B) in the C14S sub(PsaC)*PS I, C51S sub(PsaC)*PS I, C14G sub(PsaC)*PS I, and C51G sub(PsaC)*PS I mutants and less than 40% in the C14A sub(PsaC)*PS I and C51A sub(PsaC)*PS I mutants, compared with similar 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 sub(6)-NADP super(+) reductase activity, indicating lower rates in the alanine mutants. It is proposed that the chemical rescue of a [4Fe-4S] cluster with a recruited external thiolate at the modified site allows the mutant PsaC proteins to rebind to PS I and to function in forward electron transfer.