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Zymographic differentiation of [NiFe]-Hydrogenases 1, 2 and 3 of... » Isaúde
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BMC microbiology [electronic resource]
2012-07-07 03:29:09

Zymographic differentiation of [NiFe]-Hydrogenases 1, 2 and 3 of Escherichia coli K-12

Descrição: Background:When grown under anaerobic conditions, Escherichia coli K-12 is able to synthesize threeactive [NiFe]-hydrogenases (Hyd1-3). Two of these hydrogenases are respiratory enzymescatalysing hydrogen oxidation, whereby Hyd-1 is oxygen-tolerant and Hyd-2 is considered astandard oxygen-sensitive hydrogenase. Hyd-3, together with formate dehydrogenase H(Fdh-H), forms the formate hydrogenlyase (FHL) complex, which is responsible for H2evolution by intact cells. Hydrogen oxidation activity can be assayed for all threehydrogenases using benzyl viologen (BV; Eo\' = -360 mV) as an artificial electron acceptor;however ascribing activities to specific isoenzymes is not trivial. Previously, an in-gel assaycould differentiate Hyd-1 and Hyd-2, while Hyd-3 had long been considered too unstable tobe visualized on such native gels. This study identifies conditions allowing differentiation ofall three enzymes using simple in-gel zymographic assays.Results:Using a modified in-gel assay hydrogen-dependent BV reduction catalyzed by Hyd-3 hasbeen described for the first time. High hydrogen concentrations facilitated visualization of Hyd-3 activity. The activity was membrane-associated and although not essential forvisualization of Hyd-3, the activity was maximal in the presence of a functional Fdh-Henzyme. Furthermore, through the use of nitroblue tetrazolium (NBT; Eo\' = -80 mV) it wasdemonstrated that Hyd-1 reduces this redox dye in a hydrogen-dependent manner, whileneither Hyd-2 nor Hyd-3 could couple hydrogen oxidation to NBT reduction. Hydrogendependentreduction of NBT was also catalysed by an oxygen-sensitive variant of Hyd-1 thathad a supernumerary cysteine residue at position 19 of the small subunit substituted forglycine. This finding suggests that tolerance toward oxygen is not the main determinant thatgoverns electron donation to more redox-positive electron acceptors such as NBT.Conclusions:The utilization of particular electron acceptors at different hydrogen concentrations and redoxpotentials correlates with the known physiological functions of the respective hydrogenase.The ability to rapidly distinguish between oxygen-tolerant and standard [NiFe]-hydrogenasesprovides a facile new screen for the discovery of novel enzymes. A reliable assay for Hyd-3will reinvigorate studies on the characterisation of the hydrogen-evolving FHL complex.

Identificador: doi:10.1186/1471-2180-12-134
Volume: 0
Página: 1 a

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