Abstract
The gamma-proteobacterium
Shewanella oneidensis
harbours one [NiFe]- and one [FeFe]-hydrogenase. This paper presents the first biochemical characterisation of
So
HydAB, a M2(D)-type [FeFe]-hydrogenase consisting of a small subunit featuring a twin-arginine-targeting motif (TAT) and a large subunit harbouring the active site cofactor and two accessory iron-sulfur clusters. There are three main reasons to investigate this enzyme. Firstly,
S. oneidensis
is a highly studied, metabolically complex, and widely utilised organism, especially in the context of wastewater treatment, CO
2
conversion, and microbial fuel cells, including H
2
-producing systems. Advanced understanding of its hydrogen metabolism can improve biotechnological applications. Secondly,
S. oneidensis
has been a mainstay of hydrogenase research for decades as its enzymes HydE, F and G are the most widely studied and utilised maturases - enzymes assembling the active site cofactor of [FeFe]-hydrogenases. Lastly, while enzymes like
Nd
HydAB from
Nitratidesulfovibrio vulgaris
(formerly
Dd
HydAB,
Desulfovibrio vulgaris
) with high similarity to
So
HydAB have been characterised extensively in the past, the metabolic context of the enzyme in
Shewanella
is different from
Nitratidesulfovibrio
. While
Nd
HydAB receives electrons from ferredoxin,
So
HydAB accepts electrons from the menaquinone pool of the outer membrane while being a soluble enzyme in the periplasm. This is facilitated by interaction with a membrane-integrated cytochrome b transcribed from the same operon. Oxidation of the menaquinone pool drives oxidation of formate toward CO
2
via formate dehydrogenase. This study therefore lays the groundwork for understanding H
2
-dependent CO
2
turnover, a new type of metabolic niche for [FeFe]-hydrogenases, while simultaneously improving our understanding of a widely used platform organism.