Send to

Choose Destination
Biochemistry. 1999 Oct 26;38(43):14330-7.

Amino acid replacements at the H2-activating site of the NAD-reducing hydrogenase from Alcaligenes eutrophus.

Author information

Institut für Biologie, Humboldt-Universität zu Berlin, Germany.


The role of amino acid residues in the H(2)-activating subunit (HoxH) of the NAD-reducing hydrogenase (SH) from Alcaligenes eutrophus has been investigated by site-directed mutagenesis. Conserved residues in the N-terminal L1 (RGxE) and L2 (RxCGxCx(3)H) and the C-terminal L5 (DPCx(2)Cx(2)H/R) motifs of the active site-harboring subunit were chosen as targets. Crystal structure analysis of the [NiFe] hydrogenase from Desulfovibrio gigas uncovered two pairs of cysteines (motifs L2 and L5) as coordinating ligands of Ni and Fe. Glutamate (L1) and histidine residues (L2 and L5) were proposed as being involved in proton transfer [Volbeda, A., Charon, M.-H., Piras, C., Hatchikian, E. C., Frey, M., and Fontecilla Camps, J. C. (1995) Nature 373, 580-587]. The A. eutrophus mutant proteins fell into three classes. (i) Replacement of the putative four metal-binding cysteines with serine led to the loss of H(2) reactivity and blocked the assembly of the holoenzyme. Exchange of Cys62, Cys65, or Cys458 was accompanied by the failure of the HoxH subunit to incorporate nickel, supporting the essential function of these residues in the formation of the active site. Although the fourth mutant of this class (HoxH[C461S]) exhibited nickel binding, the modified protein was catalytically inactive and unable to oligomerize. (ii) Mutations in residues possibly involved in proton transfer (HoxH[E43V], HoxH[H69L], and HoxH[H464L]) yielded Ni-containing proteins with residual low levels of hydrogenase activity. (iii) The most promising mutant protein (HoxH[R40L]), which was identified as a metal-containing tetrametric enzyme, was completely devoid of H(2)-dependent oxidoreductase activity but exhibited a remarkably high level of D(2)-H(+) exchange activity. These characteristics are compatible with the interpretation of a functional proton transfer uncoupled from the flow of electrons.

[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for American Chemical Society
Loading ...
Support Center