STUDY OF THE IRON SULFUR CLUSTER AND POLYPEPTIDE COMPOSITION OF THE PHOTOSYNTHETIC REACTION CENTER FROM HELIOBACTERIUM MODESTICALDUM
Open Access
- Author:
- Heinnickel, Mark Lewis
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- January 08, 2007
- Committee Members:
- John H Golbeck, Committee Chair/Co-Chair
Squire J Booker, Committee Member
Joseph M Bollinger Jr., Committee Member
Donald Ashley Bryant, Committee Member
Dr Juliette Lecomte, Committee Member - Keywords:
- Heliobacterium
iron sulfur clusters
EPR
photosynthesis
anaerobic - Abstract:
- The photosynthetic reaction center from Heliobacterium modesticaldum (HbRC) was studied using a variety of biochemical and biophysical techniques. The purpose of the work was to obtain a better understanding of the electron transfer cofactors and polypeptides that comprise the HbRC. The major findings include: (1) the HbRC loosely binds a ferredoxin-like protein that harbors two iron-sulfur clusters. This protein, which dissociates from the HbRC when it is passed over an anion exchange resin, was named PshB. HbRC complexes purified in this manner are henceforth referred to as HbRC cores. PshB can also be removed from HbRC complexes at high ionic strength and purified by passage over an ultrafiltration membrane. PshB was shown to restore the light-induced EPR signal of [FA/FB]- (visible at low temperatures), and the characteristic flash-induced kinetic decay of P798+ when recombined with HbRC cores. (2) HbRC cores bind an interpolypeptide iron sulfur cluster termed FX. When HbRC cores are incubated with sodium dithionite in the presence of light, FX undergoes a one-electron reduction. Mossbauer and EPR data indicate the reduced cluster has a ground spin state of S=3/2. (3) Analysis of the non-heme iron content shows that HbRC cores bind 21.1 ± 1.1 Bchl g/P798. (4) The PshB protein was cloned from H. modesticaldum using knowledge of the N-terminal sequence of Fd2, a ferredoxin that had been isolated previously from Heliobacillis mobilis. When Fd2 is overexpressed in Escherichia coli with a His affinity tag and reconstituted with iron and sulfide, the holoprotein was found to co-purify with HbRC cores when subject to G-75 gel filtration chromatography and Ni-affinity chromatography. Similar to PshB from H. modesticaldum, the overexpressed Fd2 holoprotein restores the light-induced EPR signal of [FA/FB]- and the characteristic flash-induced kinetic decay of P798+ when recombined with HbRC cores. This work indicates the function of Fd2 is now properly assigned: instead of representing a soluble ferredoxin, Fd2 is PshB and is normally bound to the HbRC complex.