CHARACTERIZATION OF FA/FB-CONTAINING PROTEINS PshBI AND PshBII IN THE PHOTOSYNTHETIC REACTION CENTER OF Heliobacterium modesticaldum

Open Access
Author:
Sun, Yili
Graduate Program:
Biochemistry, Microbiology, and Molecular Biology
Degree:
Master of Science
Document Type:
Master Thesis
Date of Defense:
May 13, 2011
Committee Members:
  • John H Golbeck, Thesis Advisor
Keywords:
  • Heliobacterium modesticaldum
  • Photosynthetic Reaction Center
Abstract:
Similar to all known Type I photosynthetic reaction centers (RCs), the homodimeric RCs found in the Heliobacteriaceae utilize three [4Fe-4S] clusters termed FX, FA and FB that function as terminal electron acceptors. However, unlike their heterodimeric Photosystem I (PS I) counterpart, in which a single, tightly-bound polypeptide, named PsaC, is responsible for ligating the FA and FB clusters, two loosely-bound proteins, termed PshBI and PshBII, function as FA/FB harboring subunits in the heliobacterial RC (HbRC). Although the recombinant PshBI and PshBII with His-tags have been proved to be capable of functioning as electron acceptors, untagged PshBI and PshBII are needed to understand their 3-dimensional structures and protein interactions. In this study, reverse transcription PCR and western blot analysis have been carried out to study the transcription and translation of PshBI and PshBII in Heliobacterium modesticaldum. PshBI and PshBII have been over-expressed in Eschericia coli with a cleavable thioredoxin tag. After enterokinase cleavage, only one additional alanine remains on the N-termini of the two proteins. Untagged PshBI and PshBII have been reconstituted with inorganic iron and sulfide. Optical and low temperature X-band EPR studies indicate that untagged PshBI and PshBII harbor two [4Fe-4S] clusters. Room temperature flash-induced charge recombination kinetics and low temperature light-induced EPR spectroscopic studies indicate both untagged PshBI and PshBII are able to accept electron from FX in HbRC cores. The long lifetime kinetic phase observed in the charge recombination kinetics suggests that PshBI and PshBII might be mobile or semi-mobile subunits. Pull-down assays have been carried out using PshBI-His and PshBII-His holoproteins as bait. Several potential proteins that interact with PshBI and PshBII in H. modesticaldum have been captured and identified by mass spectrometry.