Site-directed mutagenesis of the PsaC subunit of photosystem I. F(b) is the cluster interacting with soluble ferredoxin.
The two [4Fe-4S] clusters F(A) and F(B) are the terminal electron acceptors of photosystem I (PSI) that are bound by the stromal subunit PsaC. Soluble ferredoxin (Fd) binds to PSI via electrostatic interactions and is reduced by the outermost iron-sulfur cluster of PsaC. We have generated six site-directed mutants of the green alga Chlamydomonas reinhardtii in which residues located close to the iron-sulfur clusters of PsaC are changed. The acidic residues Asp(9) and Glu(46), which are located one residue upstream of the first cysteine liganding cluster F(B) and F(A), respectively, were changed to a neutral or a basic amino acid. Although Fd reduction is not affected by the E46Q and E46K mutations, a slight increase of Fd affinity (from 1.3- to 2-fold) was observed by flash absorption spectroscopy for the D9N and D9K mutant PSI complexes. In the FA(2) triple mutant (V49I/K52T/R53Q), modification of residues located next to the F(A) cluster leads to partial destabilization of the PSI complex. The electron paramagnetic resonance properties of cluster F(A) are affected, and a 3-fold decrease of Fd affinity is observed. The introduction of positively charged residues close to the F(B) cluster in the FB(1) triple mutant (I12V/T15K/Q16R) results in a 60-fold increase of Fd affinity as measured by flash absorption spectroscopy and a larger amount of PsaC-Fd cross-linking product. The first-order kinetics are similar to wild type kinetics (two phases with t((1)/(2)) of <1 and approximately 4.5 microseconds) for all mutants except FB(1), where Fd reduction is almost monophasic with t((1)/(2)) < 1 microseconds. These data indicate that F(B) is the cluster interacting with Fd and therefore the outermost iron-sulfur cluster of PSI.
Fischer
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Departments of Molecular Biology and Plant Biology, University of Geneva, 30 quai Ernest-Ansermet, 1211 Geneva, Switzerland.
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Rochaix
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D000818:Animals; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000378:metabolism; D003080:Cold Temperature; D003432:Cross-Linking Reagents / Q000737:chemistry; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D005288:Ferredoxins / Q000378:metabolism*; D008565:Membrane Proteins; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D045331:Photosystem I Protein Complex; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012995:Solubility
D003432:Cross-Linking Reagents; D017931:DNA Primers; D005288:Ferredoxins; D008565:Membrane Proteins; D045331:Photosystem I Protein Complex; D011506:Proteins; C089909:photosystem I, psaB subunit