E. BURTON, University of Manitoba D. WONG, University of Manitoba R.R.M. SPARLING, University of Manitoba
Title
Phenazine-dependent F420H2
dehydrogenase activity is located on the
F420-reducing hydrogenase of
Methanospirillum hungatei strain GP1 and
Methanosarcina barkeri strain Fusaro
Abstract Text
We have detected a phenazine-dependent F420H2 oxidizing activity in the cell-free extract (CFE) of the hydrogenotrophic methanogen Methanospirillum (Msp.) hungatei strain GP1. Metal chelate affinity chromatography (MCAC) was used to enrich the phenazine-dependent F420H2 dehydrogenase activity. This activity co-eluted with an F420-reducing hydrogenase activity. Fractions containing dehydrogenase activity were pooled, concentrated and loaded onto a 3-20% native gradient gel. Protein bands were cut and eluted from the gel and selected for dehydrogenase activity, yielding a single pure protein containing both F420H2 dehydrogenase and F420-reducing hydrogenase activities. SDS-PAGE revealed a polypeptide pattern identical to that of the purified F420-reducing hydrogenase from Msp.hungatei. This protein is distinct from the F420H2 dehydrogenase purified from the methylotrophic methanogen Methanosarcina (Ms.) mazei Gö1, which through sequence homology is related to the NADH dehydrogenases of respiratory chains (i.e. complex I of eukarya).
A search through the genome of the methylotroph Ms.barkeri strain Fusaro using the F420H2 dehydrogenase sequence from Ms.mazei Gö1 did not reveal the presence of a distinct F420H2 dehydrogenase, although the activity is present in the CFE. To establish the source of the phenazine-dependent F420H2 dehydrogenase activity, the technique used to purify the F420H2 dehydrogenase activity from Msp.hungatei was applied to Ms.barkeri. The purified protein from Ms.barkeri also possesses F420H2 dehydrogenase and F420-reducing hydrogenase activities, with an SDS PAGE polypeptide pattern nearly identical to that of the known Ms.barkeri F420-reducing hydrogenase. Kinetics studies show that the phenazine-dependent dehydrogenase activity follows simple Michaelis-Menten kinetics and is competitively inhibited by diphenyleneiodonium (DPI) chloride; the hydrogenase activities of the protein were not affected by DPI. Methylviologen (with metronidazole) and FAD were also used as electron acceptors for the dehydrogenase activity, while NADP+ was not reduced.
The results of this study indicate that the previously characterized F420-reducing hydrogenase from both Ms.barkeri and Msp.hungatei are also capable of phenazine-dependent F420 oxidation. The physiological significance of this activity is unclear.
Untitled 1
CSM-SCM Secretariat 17 Dossetter Way
Ottawa, ON K1G 4S3
Canada
