A Study of the Structure and Metabolic Processes of a Novel Membrane Cytochrome in an Extreme Microbial Community

Wong, S E ; Jeans, C ; Thelen, M P

Lawrence Livermore National Laboratory

关键词: Cytochromes;    Membrane Proteins;    Membranes;    Enzyme Immunoassay;    Water Resources;   

DOI  :  10.2172/894351
RP-ID  :  UCRL-TR-224396
RP-ID  :  W-7405-ENG-48
RP-ID  :  894351

美国|英语

来源: UNT Digital Library

【 摘 要 】

The action of iron oxidizing microbes can generate acid mine drainage (AMD), characterized by acidic, toxic metal-tainted water that pollutes various water resources. The acidophilic biofilm community populating the Richmond mine, a pyrite (FeS{sub 2}) deposit in Northern California, is a key component of the oxidation of Fe(II) as well as subsequent pyrite dissolution. These natural biofilms contain many novel proteins that are being studied in order to understand how these microbes oxidize iron. The focus of this study is on the structure and characteristics of one novel, abundant outer membrane protein, cytochrome 572 (Cyt{sub 572}), which is perhaps important to the function of the entire community. To detect and study this cytochrome, monoclonal antibodies (mAb) were produced and screened for specificity to Cyt{sub 572}, both purified and membrane-bound. This was accomplished using enzyme linked immunosorbent assay (ELISA) and western blot analysis. Using western blotting, the presence of three high molecular weight bands at positions of dimer, trimer and tetramer corroborate chromatographic results that Cyt{sub 572} is a tetramer. Immunoprecipitation was used to detect a Cyt{sub 572} specific multiprotein complex, and these experiments are in progress. Apart from its novel amino acid sequence, Cyt{sub 572} binds to a heme group that exhibits unique spectral properties. To characterize the heme further, several biochemical methods were used to examine the purified cytochrome. Ethidium bromide was used in a novel way to detect proteins containing heme. The smallest heme-binding polypeptide fragment, about 23kDa, was identified by gel electrophoresis after proteolytic digestion of purified Cyt{sub 572}. The inability of these enzyme digests to completely degrade the protein reveals a secondary structure protective mechanism surrounding the heme group. This is perhaps associated with biofilm membrane proteins like Cyt{sub 572} that are in contact with an extremely acidic environment. Heme-protein interactions and higher order protein complexes have likely been selected for stability and the efficient transfer of electrons from Fe(II) to cells for energy, an essential function in the biofilm community.