SlyD-dependent nickel delivery limits maturation of [NiFe]-hydrogenases in late-stationary phase Escherichia coli cells

Abstract

Fermentatively growing Escherichia coli cells have three active [NiFe]-hydrogenases (Hyd), two of which, Hyd-1 and Hyd-2, contribute to H2 oxidation while Hyd-3 couples formate oxidation to H2 evolution. Biosynthesis of all Hyd involves the insertion of a Fe(CN)2CO group and a subsequent insertion of nickel ions through the HypA/HybF, HypB and SlyD proteins. With high nickel concentrations the presence of none of these proteins is required, but under normal growth conditions and during late stationary growth SlyD is important for hydrogenase activities. The slyD mutation reduced H2 production during exponential phase growth by about 50%. Assaying stationary phase grown cells for the coupling of Hyd activity to the respiratory chain or formate-dependent H2 evolution showed that SlyD is essential for both H2 evolution and H2 oxidation. Although introduction of plasmid-coded slyD resulted in an overall decrease of Hyd-2 polypeptides in slyD and hypA slyD mutants, processing and dye-reducing activity of the Hyd-2 enzyme was nevertheless restored. Similarly, introduction of the slyD plasmid restored only some H2 evolution in the slyD mutant while Hyd-3 polypeptides and dye-reducing activity were fully restored. Taken together, these results indicate an essential role for SlyD in the generation of the fully cofactor-equipped hydrogenase large subunits in the stationary phase where the level of each Hyd enzyme is finely tuned by SlyD for optimal enzyme activity.

Nickel Metalloregulators and Chaperones

“…SlyD is important for [NiFe] hydrogenase maturation [16] and is known to form a complex with HypB, [15,129,154,155]. However, the details of the interaction between SlyD and HypB still need to be determined.…”

“…However, in E. coli the isomerase function of SlyD is nonessential in [NiFe]-hydrogenase maturation [129]. In E.coli it was determined that SlyD is important for [NiFe]-hydrogenase activity as a slyD mutation resulted in a 50% reduction in H 2 production in cultures during exponential phase growth [16]. E. coli SlyD has an unstructured C-terminal metal-binding tail (Figure 11), which is rich in metal binding residues: histidines, cysteines, aspartates, and glutamates [17].…”

“…The large subunit contains a complex NiFe(CN) 2 CO center at the active site and the small subunit contains up to three iron sulfur clusters [12,13]. The assembly of [NiFe]-hydrogenase 3 is dependent on the hyp (hydrogenase pleiotropy) genes hypABCDEF and slyD genes ( Figure 1) [9,[14][15][16][17]. For the assembly of hydrogenases 1 and 2, HypA and HypC are replaced by HybF and HybG [18].…”

A whole-cell, high-throughput hydrogenase assay to identify factors that modulate [NiFe]-hydrogenase activity

“…After confirming the decrease in whole-cell hydrogenase activity, we performed a secondary hydrogenase assay that measures the hydrogen-depandant reduction of benzyl viologen in crude cell lysates. This assay was performed in an atmosphere containing 3-5% H 2 and bulk N 2 and is widely used to measure the total hydrogenase activity independent of other cell machinery such as a functioning formate dehydrogenase (5,7,9,42). Using this secondary screen, we were able to differentiate between strains that had a low whole-cell response due to a deficient hydrogen oxidation activity versus other factors such as faulty formate dehydrogenase or substrate accessibility (Fig.…”

Reasons for Thiomicrospira crunogena's recalcitrance towards previous attempts to detect its hydrogen consumption ability

“…The enzyme consists of an o'β-heterodimer where the o'-subunit (large) houses the NiFe catalytic site and the β (small) subunit hosts the Fe-S clusters for electron transport. Ni metabolism is strongly linked to the biosynthesis of [NiFe]hydrogenases on the transcriptional level and is essential for assembling and maturing an active enzyme (Peters et al, 2014;Pinske et al, 2015). Likewise, Fe has been demonstrated to be pivotal for the biosynthesis and assembly of [NiFe]-hydrogenases (Pinske and Sawers, 2014).…”