HoxE—a subunit specific for the pentameric bidirectional hydrogenase complex (HoxEFUYH) of cyanobacteria

Abstract

NAD(P)(+)-reducing hydrogenases have been described to be composed of a diaphorase (HoxFU) and a hydrogenase (HoxYH) moiety. This study presents for the first time experimental evidence that in cyanobacteria, a fifth subunit, HoxE, is part of this bidirectional hydrogenase. HoxE exhibits sequence identities to NuoE of respiratory complex I of Escherichia coli. The subunit composition of the cyanobacterial bidirectional hydrogenase has been investigated. The oxygen labile enzyme complex was purified to close homogeneity under anaerobic conditions from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 6301. The 647-fold and 1290-fold enriched purified enzyme has a specific activity of 46 micromol H(2) evolved (min mg protein)(-1) and 15 micromol H(2) evolved (min mg protein)(-1), respectively. H(2)-evolution of the purified enzyme of S. sp. PCC 6803 is highest at 60 degrees C and pH 6.3. Immunoblot experiments, using a polyclonal anti-HoxE antibody, demonstrate that HoxE co-purifies with the hydrogenase activity in S. sp. PCC 6301. SDS-PAGE gels of the purified enzymes revealed six proteins, which were partially sequenced and identified, besides one nonhydrogenase component, as HoxF, HoxU, HoxY, HoxH and, remarkably, HoxE. The molecular weight of the native protein (375 kDa) indicates a dimeric assembly of the enzyme complex, Hox(EFUYH)(2).

The Bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803 Is Reduced by Flavodoxin and Ferredoxin and Is Essential under Mixotrophic, Nitrate-limiting Conditions

“…The first evidence that NAD(P)H is able to induce hydrogen production came from measurements in cell-free extracts (11) which was confirmed in other cell-free extracts later on (3,12,13). Attempts to reduce the purified hydrogenase with NAD(P)H either failed (8,9) or resulted in hydrogen production at low rates (14,15) whereas addition of reduced MV induced hydrogen evolution at high rates.…”

“…Therefore, activity measurements with redox partners on the purified enzyme need to be interpreted cautiously concerning statements about electron transport processes in vivo as long as the integrity of the enzyme was not verified. As the bidirectional hydrogenase might be membrane-bound with HoxE anchoring the enzyme, it might even be impossible to isolate this enzyme in its native form (14,20,21). To the best of our knowledge, reduced ferredoxin was only tested once as electron donor in an essay with a purified hydrogenase, where it was shown to be ineffective to induce H 2 production (14).…”

Genomic and in situ investigations of the novel uncultured Chloroflexi associated with 0092 morphotype filamentous bulking in activated sludge

“…The putative fdhH gene is co-located with that for a probable formate transporter (FocA) (CFX0092_v1_a0448), supporting possible utilization of exogenous sources of formate, which are produced by other fermentative activated sludge bacteria under anaerobic conditions . The additional putative cytoplasmic Hox-like bi-directional hydrogenase (Schmitz et al, 2002) was detected in the Cfx-K genome, which may act as an electron valve; either oxidizing excess NADH that is generated during fermentation, or by providing reducing power for anabolic pathways when operating in reverse.…”

Molecular Biology of Microbial Hydrogenases

“…The fi rst NAD-dependent, tetrameric [NiFe]-H 2 ase was isolated from R. eutropha (A. eutrophus) (Schneider and Schlegel, 1976;Tran-Betcke et al, 1990); homologous enzymes were later discovered in cyanobacteria (Schmitz et al, 1995(Schmitz et al, , 2002Appel and Schulz, 1996) and recently in the photosynthetic bacterium T. roseopersicina (Rákhely et al, 2004). These bidirectional H 2 ases are composed of two moieties: the heterodimer [NiFe]-H 2 ase moiety encoded by the hoxY and hoxH genes, and the diaphorase moiety, encoded by the hoxU, hoxF (and also hoxE in cyanobacteria and in T. roseopersicina) genes, which is homologous to subunits of complex I of the mitochondrial and bacterial respiratory chains and contains NAD(P), FMN and Fe-S binding sites (Figure 2) (reviewed by Friedrich and Schwartz, 1993;Appel and Schulz, 1998;2004;Tamagnini et al, 2002).…”

“…Subunit NuoL, (or NuoM, or NuoN, which apparently evolved by triplication of an ancestral gene related to bacterial H + /K + antiporters (Fearnley and Walker, 1992;Friedrich and Weiss, 1997) (Appel and Schulz, 1996;Schmitz et al 2002), E. coli H 2 ase-3 (HycCDEFG) (Sauter et al, 1992) and Methanosarcina barkeri (EchABCDEF) (Künkel et al, 1998) (Adapted from Dupuis et al, 2001;Holt et al, 2003;Hedderich 2004 164 Vignais and Colbeau would have provided the transmembrane channel required to complete the proton (or Na + ) pump (Dupuis et al, 2001;Mathiesen and Hagerhall, 2002;Hedderich, 2004).…”