Role of HoxE subunit in Synechocystis PCC6803 hydrogenase

Abstract

Cyanobacterial NAD(P) + -reducing reversible hydrogenases comprise five subunits. Four of them (HoxF, HoxU, HoxY, and HoxH) are also found in the well-described related enzyme from Ralstonia eutropha. The fifth one (HoxE) is not encoded in the R. eutropha genome, but shares homology with the N-terminal part of R. eutropha HoxF. However, in cyanobacteria, HoxE contains a 2Fe-2S cluster-binding motif that is not found in the related R. eutropha sequence. In order to obtain some insights into the role of HoxE in cyanobacteria, we deleted this subunit in Synechocystis PCC6803. Three types of interaction of the cyanobacterial hydrogenase with pyridine nucleotides were tested: (a) reductive activation of the NiFe site, for which NADPH was found to be more efficient than NADH; (b) H 2 production, for which NADH appeared to be a more efficient electron donor than NADPH; and (c) H 2 oxidation, for which NAD + was a much better electron acceptor than NADP + . Upon hoxE deletion, the Synechocystis hydrogenase active site remained functional with artificial electron donors or acceptors, but the enzyme became unable to catalyze H 2 production or uptake with NADH ⁄ NAD + . However, activation of the electron transfer-independent H ⁄ D exchange reaction by NADPH was still observed in the absence of HoxE, whereas activation of this reaction by NADH was lost. These data suggest different mechanisms for diaphorase-mediated electron donation and catalytic site activation in cyanobacterial hydrogenase.

Cyanobacterial Hydrogenases and Hydrogen Metabolism Revisited: Recent Progress and Future Prospects

“…The authors discussed that under anoxia, the reduced NADPH pools may be consumed by the bidirectional Hox hydrogenase [35]. Contrarily, studies on HoxE deletion strain suggested better performance of NADH over NADPH, as electron donors to the bidirectional Hox hydrogenase in Synechocystis [78]. H/D exchange rates conducted on soluble wild type cell extracts indicated that NADH was a much better electron donor compared to NADPH, however for the HoxE deletion mutant, supplementation of the soluble extract with NADPH showed higher activity.…”

“…H/D exchange rates determine the turnover of the catalytic site independently of the electron transfer. This led the authors to conclude that whereas NADH-mediated interaction occurred via the diaphorase moiety, the NADPH interaction occurred through an alternate mechanism [78]. Although it was generally accepted that the pyridine nucleotides were involved in transport of electrons to the Hox hydrogenase, there was general confusion regarding the nature; NADPH or NADH.…”

“…Moreover, Gutekunst et al . [16] suggested that assays in which NAD(P)H was regarded as the electron donor used high concentrations between 12 µM to 0.3 mM [16,35,78]. Such high concentrations cannot be accounted for in the living cell [16].…”

“…Such high concentrations cannot be accounted for in the living cell [16]. In addition, lately it was shown that NADPH was a poor electron donor as compared to NADH [16,78]. This was in contradiction to the earlier propagated role of the bidirectional Hox hydrogenase as an electron sink for the disposal of surplus electrons coming from PSI via ferredoxin, FNR and NADPH.…”

“…In view of the above, alternate molecules that could serve as redox acceptors or donors to the bidirectional Hox hydrogenase were surveyed [16,78]. Important limitations of the studies conducted so far appear to be lack of any in vivo analysis.…”