Modified ligands to FA and FB in photosystem I. Proposed chemical rescue of a [4Fe-4S] cluster with an external thiolate in alanine, glycine, and serine mutants of PsaC.
The FB and FA 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-FX cores, resulting in fully functional PS I complexes. The low temperature EPR spectra of the C14XPsaC.PS I complexes (where X = S, A, or G) show the photoreduction of a wild-type FA cluster and a modified FB' 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 C51SPsaC.PS I complex differs from that of the C51APsaC.PS I or C51GPsaC.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 FA and FB are simultaneously reduced. Single turnover flash studies indicate approximately 50% efficient electron transfer to FA/FB in the C14SPsaC.PS I, C51SPsaC.PS I, C14GPsaC.PS I, and C51GPsaC.PS I mutants and less than 40% in the C14APsaC.PS I and C51APsaC.PS I mutants, compared with approximately 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 PS I and to function in forward electron transfer.
Jung
Y S
YS
Department of Biochemistry, and Center for Biological Chemistry, George W. Beadle Center, University of Nebraska, Lincoln, Nebraska 68588-0664, USA. jhg5@psu.edu
Vassiliev
I R
IR
Qiao
F
F
Yang
F
F
Bryant
D A
DA
Golbeck
J H
JH
D000409:Alanine; D001426:Bacterial Proteins; D003580:Cytochromes / Q000378:metabolism; D045348:Cytochromes f; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D005998:Glycine; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045331:Photosystem I Protein Complex; D012694:Serine; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D003580:Cytochromes; D007506:Iron-Sulfur Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; C096017:PscA protein, green sulfur bacteria; D012694:Serine; D009249:NADP; D045348:Cytochromes f; D000409:Alanine; D005998:Glycine