iron sulfur cluster assembly in photosystem I of cyanobacteria: the role of SufR (SUF) and Fdx (ISC)
Open Access
- Author:
- Wang, Tao
- Graduate Program:
- Plant Physiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- April 07, 2004
- Committee Members:
- John H Golbeck, Committee Chair/Co-Chair
Donald Ashley Bryant, Committee Chair/Co-Chair
Teh Hui Kao, Committee Member
Hong Ma, Committee Member - Keywords:
- Iron-sulfur cluster
assembly
biogenesis
PS I
SufR
Fdx
cyanobacteria - Abstract:
- The assembly/biogenesis of Photosystem I is poorly understood compared with its well-documented structure and function studies. As a multi-subunit membrane pigment-protein complex, many factors affect its assembly. Among them three [4Fe-4S] clusters are functionally and structurally essential. Any single cysteine ligand change would result in reduction or complete loss of PS I complex in cyanobacteria. Fe-S cluster assembly is a complicated bioprocess involving several protein factors. So far three Fe-S cluster assembly pathways have been identified, namely NIF, ISC, and SUF. Cyanobacteria are distinguished from other organisms in regard to Fe-S assembly, as it contains four nifS-like homologs representing three possible pathways. slr0387 and sll0704 are iscS-like homologs (ISC), slr0077 is sufS-like homolog (SUF), and slr2143 does not belong to any known Fe-S cluster assembly pathway so far. To investigate which one is involved in Fe-S cluster assembly in PS I, two strategies were applied to start study on the regulation of Fe-S cluster assembly into PS I. 1), pseudorevertant screening of C14SPsaC mutant in Synechocystis sp. PCC 6803 to find regulators of Fe-S cluster assembly; 2), molecular and genetic manipulation on homologous genes encoding components of known Fe-S cluster assembly pathway. For the first strategy, sll0088 was found to be involved in the assembly of C14SPsaC mutant protein into PS I complex by pseudorevertant screening. Amino acid sequence alignment of Sll0088 homologs indicated that Sll0088 contains an HTH DNA binding domain and a unique cysteine motif CX12CX13CX14C. In almost all cyanobacterial genomes sequenced so far, sll0088-like gene is located immediately upstream of a suf gene cluster and divergently transcribed. To investigate if it encodes a transcription regulator, the sll0088-like gene in Synechococcus sp. PCC 7002 was insertionally inactivated. RT-PCR analysis showed that the four suf genes organize as an operon and their transcription was up-regulated in sll0088-like inactivation mutant. sll0088-like was thus renamed sufR (suf repressor). SufR also has other regulons, such as slr0387 (iscS homolog), which is down-regulated in sufR inactivation mutant. SufR protein was overexpressed in E. coli and purified. EPR and Mössbauer studies indicated that reconstituted SufR is a [4Fe-4S] protein. The ratio is one Fe-S cluster per dimer. Amino acid substitution study suggested that the 1st, 2nd and 4th cysteine residues in the cysteine motif provide ligands to Fe-S cluster. Replacement of the 3rd one with either serine or glycine resulted in neglectable EPR signal changes and thus this cysteine may not participate in Fe-S cluster ligation. The reconstituted Fe-S cluster is oxygen sensitive. Physiologically, SufR protein may use [4Fe-4S] cluster as a sensor to respond to oxidative stress and iron deficiency, as under both conditions the suf genes were derepressed. The SufR protein was also found to be distributed into two compartments by immunoblot analysis, the majority exists in the cytosol, about one third associates with the thylakoid membrane, suggesting that SufR might be multi-functional. Function of the membrane-bound SufR remains unknown. For the second strategy, we focused on an fdx (ISC) homolog, slr0148 in Synechocystis sp. PCC 6803. It was identified to be the fdx homolog of ISC Fe-S cluster assembly pathway based on amino acid sequence similarity and the finding that recombinant protein can coordinate a [2Fe-2S] cluster. To further demonstrate that slr0148 is the Fdx component of ISC machinery, the slr0148 gene was insertionally inactivated and growth phenotypes of slr0148 mutant was compared with those of slr0387 and sll0704 inactivation mutants under standard, high light, low temperature, and Fe limitation conditions. Their transcription under these growth conditions and oxidative stress condition, together with that of the other two nifS-like gene was also investigated by RT-PCR. We found that the four nifS-like genes were differentially expressed under various conditions, clearly indicating that each NifS-like or in combination played a leading role under certain stress conditions. slr0148 was reported to be involved in a large gene cluster spanning from slr0144 through slr0152 by microarray analyses. Our RT-PCR data indicated that this gene cluster forms a large operon spanning from slr0144 to slr0151. We further confirmed the microarray finding that genes involved in this operon were down-regulated in response to high light, cold shock, H2O2 treatment, and Fe starvation, although there are a few variations in regard to certain genes under some conditions. Functions of the overall gene cluster might be related to photosystem I assembly/turnover. Based on these studies, we concluded that SUF plays a major and essential in Fe-S cluster assembly in cyanobacteria, and ISC is only a backup pathway.