Iron is essential to almost all life and organisms use iron for a variety of vital functions. Rhodopseudomonas palustris strain TIE-1 obtains energy by oxidizing iron and recently, the proteins required for iron oxidation were identified. One of these proteins, PioB, is proposed here to be an iron transport protein because 1) the predicted porin-like structure of PioB is similar to other iron transport proteins and 2) of the different protein required for iron oxidation, PioB is the only protein predicted to be in the outer membrane. PioB is also proposed to represent a novel class of iron transport proteins because it lacks several of the conserved sequence motifs found in other porin-like iron transport proteins. Furthermore, PioB is required in conditions of high extracellular iron while the other structurally similar proteins are only essential in iron deplete conditions. The goal of this study is to elucidate the function and mechanisms of PioB. The first specific aim (1) focuses on determining the subcellular locations of PioB and the other proteins required for iron oxidation using subcellular fractionation and western blot analysis on wild type and PioB null mutant R. palustris. The goal of the second specific aim (2) is to test the hypothesis that PioB is an iron transport protein by comparing iron content in cells expressing PioB with PioB null cells. Mutagenic studies are also proposed to tease out important functional residues and structural elements of the protein. The third specific aim (3) proposes to develop a purification protocol of the membrane bound PioB protein for further in vitro characterization. The tools of microbiology, molecular biology, and bioinorganic chemistry will be employed to understand further the machinery of iron regulation utilized by biological systems. Iron is one of the most important heavy metals utilized in biological systems. Although iron is required, organisms must strike a fine balance between the toxic extremes of iron deficiency and iron over- accumulation and medical disorders exist on both ends of this spectrum. PioB is a putative novel iron transport protein and understanding the functional and structural aspects of this protein will provide important insights to the general mechanisms organisms use to regulate iron. ? ? ?
