Identification and isolation of the polyferredoxin from Methanobacterium thermoautotrophicum strain delta H.

Abstract

Sequencing the genes encoding the methyl viologen-reducing hydrogenase, cloned from Methanobacterium thermoautotrophicum strain delta H and Methanothermus fervidus, revealed the presence of tightly linked genes, designated mvhB, which were predicted to encode proteins containing six tandemly arranged bacterial ferrodoxin-like domains. A lacZ-mvhB gene fusion has been constructed and expressed in Escherichia coli. Rabbit antibodies raised against the fusion polypeptide purified from E. coli have been used to identify and isolate the polyferrodoxin from Mb. thermoautotrophicum strain delta H. The polyferredoxin accumulates in cells of the methanogen during exponential growth but decreases rapidly on entry into stationary phase. It is not processed into monoferredoxins and is located primarily in the soluble fraction of cell lysates of Mb. thermoautotrophicum. Metronidazole reduction by crude extracts of Mb. thermoautotrophicum strain delta H cells, dependent on the presence of hydrogen and the heterodisulfide CoM-S-S-HTP [formed from the two coenzymes 2-mercaptoethanesulfonic acid (coenzyme M, HS-CoM) and N-(7-mercaptoheptanoyl)threonine O3-phosphate (HS-HTP)], was not inhibited by the antibodies raised against the LacZ-MvhB fusion polypeptide.