Strains of Synechocystis sp. PCC 6803 with Altered PsaC

Abstract

A psaC deletion mutant of the unicellular cyanobacterium Synechocystis sp. PCC 6803 was utilized to incorporate site-specific amino acid substitutions in the cysteine residues that ligate the FA and FB iron-sulfur clusters in Photosystem I (PS I). Cysteines 14 and 51 of PsaC were changed to aspartic acid (C14DPsaC, C51DPsaC, C14D/C51DPsaC), serine (C14SPsaC, C51SPsaC), and alanine (C14APsaC, C51APsaC), and the properties of FA and FB were characterized by electron paramagnetic resonance spectroscopy and time-resolved optical spectroscopy. The C14DPsaC-PS I and C14SPsaC-PS I complexes showed high levels of photoreduction of FA with g values of 2.045, 1. 944, and 1.852 after illumination at 15 K, but there was no evidence of reduced FB in the g = 2 region. The C51DPsaC-PS I and C51SPsaC-PS I complexes showed low levels of photoreduction of FB with g values of 2.067, 1.931, and 1.881 after illumination at 15 K, but there was no evidence of reduced FA in the g = 2 region. The presence of FB was inferred in C14DPsaC-PS I and C14SPsaC-PS I, and the presence of FA was inferred in C51DPsaC-PS I and C51SPsaC-PS I by magnetic interaction in the photoaccumulated spectra and by the equal spin concentration of the irreversible P700(+) cation generated by illumination at 77 K. Flash-induced optical absorbance changes at 298 K in the presence of a fast electron donor indicate that two electron acceptors function after FX in the four mutant PS I complexes at room temperature. These data suggest that a mixed-ligand [4Fe-4S] cluster is present in the mutant sites of C14X-PS I and C51X-PS I (where X = D or S), but that the proposed spin state of S = 3/2 renders the resonances undetectable in the g = 2 region. The C14APsaC-PS I, C51APsaC-PS I and C14D/C51DPsaC-PS I complexes show only the photoreduction of FX, consistent with the absence of PsaC. These results show that only those PsaC proteins that contain two [4Fe-4S] clusters are capable of assembling onto PS I cores in vivo.

The Eight-amino Acid Internal Loop of PSI-C Mediates Association of Low Molecular Mass Iron-Sulfur Proteins with the P700-FXCore in Photosystem I

“…We can envision two mechanisms to explain these results. Illumination of PS I at cryogenic temperatures result in reduction of F A in 76% and F B in 24% of the complexes (40). This distribution is similar to that calculated assuming an equilibrium between F B and F A with redox midpoint potentials at Ϫ580 mV and Ϫ520 mV, respectively (41).…”

Influence of the [4Fe–4S] cluster coordinating cysteines on active site maturation and catalytic properties of C. reinhardtii [FeFe]-hydrogenase

“… 23 , 31 , 40 , 41 Exchanges of the [4Fe–4S] cluster coordinating cysteines to aspartate have been shown before to be functionally tolerated in the PsaC subunit of photosystem I, as well as in the small subunit of a [NiFe]-hydrogenase. 32 , 33 By following the occupancies of the [4Fe–4S] and [2Fe] cluster of the catalytically relevant H-cluster, we have been able to gain new insights into the importance of each individual cysteine coordinating the [4Fe–4S] cluster.…”

“…One explanation is that the majority of the clusters were turned into an EPR silent state by the selected amino acid exchanges, an effect already observed for a [4Fe–4S] cluster within the PsaC subunit of photosystem I (exchange of cysteine to aspartate). 33 Furthermore, in the native protein, the [4Fe–4S] cluster coordinating cysteine 366 is exposed to the surface of the maturation channel, providing the site of covalent attachment of the [2Fe] site. 42 It was shown that upon variation of this bridging position the covalent linkage of the [2Fe] cluster to the [4Fe–4S] cluster, as well as to the protein, is no longer feasible.…”

“…In this study the [4Fe–4S] cluster coordinating cysteines in HydA1 were individually substituted with alanine, aspartate or serine creating a pool of 12 new variants. In contrast to alanine, serine 29 – 31 and aspartate 32 , 33 may serve as direct oxygenic ligands in the form of serinate or aspartate. 23 Detailed analysis of the obtained variants by UV/vis, electron paramagnetic resonance (EPR), Fourier-transform infrared (FTIR) spectroscopy, solution based activity assays, and protein-film electrochemistry offered insights into the importance of the cysteines coordinating the [4Fe–4S] cluster.…”

Structure of C42D Azotobacter vinelandii FdI

“…Solvents such as acetonitrile or dimethyl sulfoxide also induce spin mixtures in the synthetic analog [Fe 4 S 4 (SR) 4 ] 3Ϫ (R ϭ alkyl or aryl) (80). Finally, the 2[4Fe-4S] 2ϩ/ϩ PsaC protein mutants, C14D and C51D, are also known to be easily converted from S ϭ 3/2 to S ϭ 1/2 at the modified cluster upon binding to the photosystem I core complex, a situation that is likely to affect solvent accessibility (81)(82)(83)(84)(85) ϩ as the dominant species and S ϭ 1/2 and 5/2 as minor components (86). In Thauera aromatica ferredoxin, which contains cysteine only-ligated 2[4Fe-4S] 2ϩ/ϩ clusters, a spin mixture of S ϭ 3/2 and 5/2 is observed for one cluster in the reduced state (87 cluster upon exposure to oxygen.…”