The F(B) and F(A) electron acceptors 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, 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(X) cores, resulting in fully functional PSI complexes. The low temperature EPR spectra of the C14X(PsaC)·PS I complexes (where X = S, A, or G) show the photoreduction of a wild-type F(A) cluster and a modified F(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 β-mercaptoethanol has likely been recruited to supply the requisite ligand to the [4Fe-4S] cluster. The EPR spectrum 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 additional set of resonances, which may be derived from the serine oxygen- ligated cluster. In all other mutant PSI complexes, a wild-type spin-coupled interaction spectrum appears when F(A) and F(B) are simultaneously reduced. Single turnover flash studies indicate ~50% efficient electron transfer to F(A)/F(B) in the C14S(PsaC)·PS I, C51S(PsaC)·PS I, C14G(PsaC)·PS I, and C51G(PsaC)·PS I mutants and less than 40% in the C14A(PsaC)·PS I and C51A(PsaC)·PS I mutants, compared with ~76% in the PS I core reconstructed with wild-type PsaC. These data are consistent with the measurements of the rates of cytochrome c6-NADP+ 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 PSI and to function in forward electron transfer.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology