Characterization of cytochromes from Methanosarcina strain Göl and their involvement in electron transport during growth on methanol

Abstract

Methanosarcina strain Gol was tested for the presence of cytochromes. Low-temperature spectroscopy, hemochrome derivative spectroscopy, and redox titration revealed the presence of two b-type (b559 and b564) and two c-type (Cs47 and c552) cytochromes in membranes from Methanosarcina strain Gol. The midpoint potentials determined were Em 7 =-135 + 5 and -240 + 11 mV (b-type cytochromes) and Em 7 =-140 10 and -230 10 mV (c-type cytochromes). The protoheme IX and the heme c contents were 0.21 to 0.24 and 0.09 to 0.28 ,Imol/g of membrane protein, respectively. No cytochromes were detectable in the cytoplasmic fraction. Of various electron donors and acceptors tested, only the reduced form of coenzyme F420 (coenzyme F420H2) and the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreonine phosphate (CoM-S-S-HTP) were capable of reducing and oxidizing the cytochromes at a high rate, respectively. Addition of CoM-S-S-HTP to reduced cytochromes and subsequent low-temperature spectroscopy revealed the oxidation of cytochrome b564. On the basis of these results, we suggest that one or several cytochromes participate in the coenzyme F420H2-dependent reduction of the heterodisulfide.Methanogenic bacteria gain metabolic energy by coupling the conversion of a number of simple substrates such as H2-CO2, formate, methylamines, methanol, and acetate to the generation of ATP. The methanogenic pathways leading to the conversion of these substrates have been unraveled in the last two decades, and they involve a number of unique cofactors (37). The penultimate step of methanogenesis, the reduction of the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreonine phosphate (CoM-S-S-HTP) with either H2 or reduced coenzyme F420 (F420H2) as a reductant, was shown in Methanosarcina strain Gol to be coupled to electron transport phosphorylation (7,34 Membrane preparations. For the spectroscopic and potentiometric characterization of cytochromes, membranes were prepared under aerobic conditions. The freshly harvested cells were washed twice with 50 mM potassium phosphate buffer, pH 7.0, containing 0.2 M sucrose and 10 mM MgSO4. After suspension in 50 mM potassium phosphate buffer, pH 7.0 (KP buffer), and addition of a few crystals of DNasev the cells were lysed by passing them through an Aminco French pressure cell at 138 MPa. The resulting crude extract was centrifuged at 10,000 x g for 15 min to remove unbroken cells and cell debris. The supernatant was diluted to 150 ml with KP buffer and centrifuged at 120,000 x g for 2 h. The supernatant (cytoplasmic fraction) was carefully removed and discarded. The membrane pellet was resuspended in the same buffer and centrifuged at 10,000 x g for 15 min to pellet the residual sulfides apparently adhering to the membranes. The supernatant from this low-speed centrifugation was subsequently subjected to ultracentrifugation at 120,000 x g for 30 min. These steps (resuspension of the resulting pellet, low-speed centrifugation, and ultracentrifugation) were repeated two more times. The final m...

Analysis of the vhoGAC and vhtGAC Operons from Methanosarcina mazei Strain Gö1, Both Encoding a Membrane‐bound Hydrogenase and a Cytochrome b

“…The same effect was described for membrane preparations of acetate-grown M s barkeri by Kemner et al (1987). Additionally, a membrane-bound multienzyme complex composed of a Fa,,-nonreactive hydrogenase, a cytochrome b, and the heterodisulfide reductase was isolated (Heiden et al, 1993(Heiden et al, , 1994 reported to accept electrons from reduced F4," (Kamlage and Blaut, 1992). Cytochrome reduction by H, was reported to be comparatively slow.…”

“…strojuan et al, 1992)] contain two b-type and two c-type cytochromes, as determined by low-temperature spectroscopy, hemochrome-derivative spectroscopy and redox titrations (unpublished results). According to these studies, the cytochromes from Ms mazei C16 have essentially the same features as those from Ms mazei strain GO1 (Kamlage and Blaut, 1992). To clone the corresponding genes, two out of the four cytochromes present in membranes of methanol-grown Ms mazei C16 were purified.…”

Isolation of a cytochrome-deficient mutant strain of Sporomusa sphaeroides not capable of oxidizing methyl groups

“…Washed membrane fractions were prepared under aerobic conditions as described before (19). The redox difference spectra of washed membranes were recorded at room temperature by reducing a sample with a few grains of Na2S204 and measuring it against an air-oxidized sample with a Perkin-Elmer/ Hitachi spectrophotometer (model 556) in the double-beam mode (18). The spectral bandwidth was set to 1 nm, and the scanning speed was 30 nm/min.…”

Heme Biosynthesis in Methanosarcina barkeri via a Pathway Involving Two Methylation Reactions

“…M. barkeri is a strictly anaerobic methanogenic archaeon that is able to grow on H 2 and CO 2 , acetate, methylamines, or methanol as sole carbon and energy sources. It is a member of the order Methanosarcinales, the members of which are all known to contain cytochromes b and c and other proteins with protoheme-derived prosthetic groups (12,15). The organism also contains the nickel tetrapyrrole coenzyme F 430 (5) and the cobalt tetrapyrrole hydroxybenzimidazolyl cobamide, which is a vitamin B 12 homolog (20).…”