Structure of the membrane-bound formate hydrogenlyase complex from Escherichia coli

Abstract

The prototypical hydrogen-producing enzyme, the membrane-bound formate hydrogenlyase (FHL) complex from Escherichia coli, links formate oxidation at a molybdopterin-containing formate dehydrogenase to proton reduction at a [NiFe] hydrogenase. It is of intense interest due to its ability to efficiently produce H2 during fermentation, its reversibility, allowing H2-dependent CO2 reduction, and its evolutionary link to respiratory complex I. FHL has been studied for over a century, but its atomic structure remains unknown. Here we report cryo-EM structures of FHL in its aerobically and anaerobically isolated forms at resolutions reaching 2.6 Å. This includes well-resolved density for conserved loops linking the soluble and membrane arms believed to be essential in coupling enzymatic turnover to ion translocation across the membrane in the complex I superfamily. We evaluate possible structural determinants of the bias toward hydrogen production over its oxidation and describe an unpredicted metal-binding site near the interface of FdhF and HycF subunits that may play a role in redox-dependent regulation of FdhF interaction with the complex.

A redox‐active HybG‐HypD scaffold complex is required for optimal ATPase activity during [NiFe]‐hydrogenase maturation in Escherichia coli

“…H 2 is dependent on the HypCD-dependent synthesis and insertion of Fe(CN) 2 CO group into the large subunit of Hyd-3, the hydrogenase component of formate hydrogenlyase complex [1,19]. Together, these observations highlight the importance of cysteine thiolates in the HypCD scaffold complex and support the role of sulfur chemistry in the maturation of Hyd [1,14].…”

“…An earlier mutagenesis study demonstrated that when C69 and C72 are exchanged for alanine, the resulting E. coli strains synthesizing these variant HypD proteins failed to produce H 2 gas [ 14 ]. The ability of the formate hydrogenlyase complex to produce H 2 is dependent on the HypCD‐dependent synthesis and insertion of Fe(CN) 2 CO group into the large subunit of Hyd‐3, the hydrogenase component of formate hydrogenlyase complex [ 1 , 19 ]. Together, these observations highlight the importance of cysteine thiolates in the HypCD scaffold complex and support the role of sulfur chemistry in the maturation of Hyd [ 1 , 14 ].…”

Perspective elucidating the physiology of a microbial cell: Neidhardt's Holy Grail

“…This is also the case for Escherichia coli , which can use its conjugate base, formate, as an electron source for respiration with oxygen or nitrate as an acceptor, or during fermentation, where it is imported into the cell by the formate‐nitrite transporter channel protein, FocA (Kammel et al, 2022). In the cytoplasm, formic acid is disproportionated to CO 2 and H 2 by the membrane‐associated formate hydrogenlyase (FHL) complex (Peters & Sargent, 2023; Steinhilper et al, 2022). The benefits to the cell of taking up formate are two‐fold: first, if formic acid is imported into the cell, this helps offset acidification of the cell's immediate environment (Metcalfe et al, 2022); second, if formate, rather than formic acid, is taken up and subsequently formate plus a cytoplasmic proton are converted into gaseous H 2 plus CO 2 by the FHL complex (Peters & Sargent, 2023), then this helps offset acidification of the cytoplasm and, additionally, potentially contributes to the establishment of an ion (proton) gradient (Kammel et al, 2022).…”

FocA and its central role in fine-tuning pH homeostasis of enterobacterial formate metabolism

“…FHL-1 and FHL-2 differ in the number and function of their respective transmembrane subunits: FHL-1 has two, while FHL-2 has five integral membrane subunits [10,[12][13][14]. This indicates different roles for these subunits in ion translocation and possibly in energy conservation [3,11,14].…”

“…Both complexes are also membrane-associated and this is essential for functionality [11] and suggests an important role for the transmembrane subunits in ion translocation [10]. FHL-1 and FHL-2 differ in the number and function of their respective transmembrane subunits: FHL-1 has two, while FHL-2 has five integral membrane subunits [10,[12][13][14]. This indicates different roles for these subunits in ion translocation and possibly in energy conservation [3,11,14].…”