Structural aspects and immunolocalization of the F420-reducing and non-F420-reducing hydrogenases from Methanobacterium thermoautotrophicum Marburg

Abstract

The F420-reducing hydrogenase and the non-F420-reducing hydrogenase (EC 1.12.99.1.) were isolated from a crude extract of Methanobacterium thermoautotrophicum Marburg. Electron microscopy of the negatively stained F420-reducing hydrogenase revealed that the enzyme is a complex with a diameter of 15.6 nm. It consists of two ring-like, stacked, parallel layers each composed of three major protein masses arranged in rotational symmetry. Each of these masses appeared to be subdivided into smaller protein masses. Electron microscopy of negatively stained samples taken from intermediate steps of the purification process revealed the presence of enzyme particles bound to inside-out membrane vesicles. Linker particles of 10 to 20 kDa which mediate the attachment of the hydrogenase to the cytoplasmic membrane were seen. Immunogold labelling confirmed that the F420-reducing hydrogenase is a membrane-bound enzyme. Electron microscopy of the negatively stained purified non-F420-reducing hydrogenase revealed that the enzyme is composed of three subunits exhibiting different diameters (5, 4, and 2 to 3 nm). According to immunogold labelling experiments, approximately 70% of the non-F420-reducing hydrogenase protein molecules were located at the cell periphery; the remaining 30% were cytoplasmic. No linker particles were observed for this enzyme.The methanogenic archaebacterium Methanobacterium thermoautotrophicum Marburg (DSM 2133) grows on H2 and CO2 as sole electron, carbon, and energy sources (14). Two types of hydrogenase have been detected in numerous methanogenic bacteria, including M. thermoautotrophicum (3,7,9,10,13,21,25,27,29,36,37,48), i.e., an F420 (8-hydroxy-5-deazaflavin)-reducing hydrogenase and a non-F420-reducing hydrogenase, both of which can be assayed via the reduction of viologen dyes with H2 (16). The F420-reducing hydrogenase also reduces the physiological two-electron acceptor F420.The F420-reducing hydrogenase contains approximately 0.7 mol of nickel, 0.9 mol of flavinadenine dinucleotide, and 13 to 14 mol of nonheme iron and acid-labile sulfur per mol of alp1,yj (2,12). The non-F420-reducing hydrogenase contains nickel and iron-sulfur clusters in undetermined amounts (18).The non-F420-reducing hydrogenase is part of an H2-heterodisulfide oxidoreductase complex in the methylotrophic bacterium Methanosarcina barkeri (19) and the hydrogenotrophic bacterium M. thermoautotrophicum. In the methylotrophic organism, this complex also contains cytochrome b and is tightly membrane associated (19), whereas the complex in the hydrogenotrophic organism contains no cytochromes and is weakly membrane associated (41). In the methylotrophic organism, electrons are transferred via these cytochromes to the heterodisulfide reductase, thereby generating a proton potential (7, 8), whereas the electron carrier in the hydrogenotrophic organism remains unknown. The present communication reports on a systematic electron microscopic investigation of the F420-reducing hydrogenase and the non-F420-reducing hydrogenase isolated f...

Identification of a Novel Vinyl Reductase Gene Essential for the Biosynthesis of Monovinyl Chlorophyll in Synechocystis sp. PCC6803

“…It is also possible that additional proteins are required for its catalytic activity. Slr1923 has sequence similarity to the gene encoding the ␤ subunit of FRH in M. thermoautotrophicum (24). Three heteromeric FRH subunits (␣, ␤, and ␥) form an (␣␤␥) 8 complex in this organism.…”

Outer Membrane Targeting, Ultrastructure, and Single Molecule Localization of the Enteropathogenic Escherichia coli Type IV Pilus Secretin BfpB

“…Electron microscopic imaging was performed with a Philips EM 301 transmission electron microscope at calibrated magnifications. The resulting images were grouped into projection forms essentially as described previously (11) and processed by modified Markham rotational analysis (38,50).…”

Biochemistry of methanogenesis: a tribute to Marjory Stephenson:1998 Marjory Stephenson Prize Lecture

“…Hydrogenotrophic methanogens can contain up to four different types of hydrogenases and of each type, several isoenzymes Sorgenfrei et al, 1997a, b ;Kunkel et al, 1998 ; for topology see Braks et al, 1994) : (i) F,,,-reducing Ni/Fe-hydrogenase ; (ii) F,,,-non-reducing hydrogenase of unknown physiological electron acceptor -in Methanosarcina spp., one of the subunits from the enzyme is a cytochrome 6 (Deppenmeier, 1995 ;Deppenmeier et al, 199.5;Kumazawa et al, 1994;Kemner & Zeikus, 1994a, b) and in Methanobacterium spp., which are devoid of cytochromes, the electron accceptor is possibly either a 44 kDa polyferredoxin (Reeve et al, 1989; (Gorris & van der Drift, 1994). F,,, in methanogens is converted to inactive F,,, by adenylation and F,,, to F,,, by deadenylation in response to different growth conditions (Vermeij et al, 1994(Vermeij et al, , 1995(Vermeij et al, , 1996 (1996) (1997) ; Widdel & Frimmer (1995) See Table 2 See Table 2 Haase et al (1992) ; Abken & Deppenmeier (1997); Abken et al (1998a, b) Vaupel (1993 Hedderich et al, 1992;Nolling et al, 1995c) or a 45 kDa flavoprotein (Wasserfallen et a/., 1995;Nolling et a/., 1995c) ; (iii) Escherichia coli hydrogenase-3-type Ni/Fehydrogenase of unknown physiological electron acceptor (Kunkel et al, 1998); and (iv) a metal-free hydrogenase, the H,-forming methylenetetrahydromethanopterin dehydrogenase, which together with the F,,,-dependent methylenetetrahydromethanopterin dehydrogenase catalyses the reduction of F4,, with H, Reeve et al, 1997).…”