Abstract: The invention relates to [NiFe]-hydrogenases having an improved resistance to dioxygen, said [NiFe]-hydrogenases may be obtained by:—providing an initial polynucleotide comprising a sequence encoding a large subunit of a [NiFe]-hydrogenase, said large subunit comprising the following peptide motifs: •L1: RGXE, wherein X=L, I, F, V or M•L2: [R/K]X1C[G/R]X2C, wherein Xi is any amino acid residue, X2=L, V, I or M; L1 and L2 being separated by 16 any amino acid residues; •L3: X1X2X3X4X5X6X7X8X9X10X11X12[D/S/E], wherein X1=D, S, N or E, X2=H, D, S, N or L, X5=H, S, A, Q or W, X6=F, T, Y or G, X9=L, F, M or Y, the other Xn being any amino acid residue; •L4: D[P/I/S]CX1X2CX3X4[H/R], wherein X2=A, S, V, G or T, X1, X3 and X4 are any amino acid residue • and optionally comprising a motif LO: R[I/V/A]EG[H/D/A].—modifying said initial polynucleotide in order to substitute at least one of the residues X2 of motif L2 and Z or X4 of motif L3 and Z or X9 of motif L3 of said large subunit by a methionine.

Abstract: The invention relates to [NiFe]-hydrogenases having an improved resistance to dioxygen, said [NiFe]-hydrogenases may be obtained by:—providing an initial polynucleotide comprising a sequence encoding a large subunit of a [NiFe]-hydrogenase, said large subunit comprising the following peptide motifs: L1: RGXE, wherein X=L, I, F, V or M L2: [R/K]X1C[G/R]X2C, wherein Xi is any amino acid residue, X2=L, V, I or M; L1 and L2 being separated by 16 any amino acid residues; L3: X1X2X3X4X5X6X7X8X9X10X11X12[D/S/E], wherein X1=D, S, N or E, X2=H, D, S, N or L, X5=H, S, A, Q or W, X6=F, T, Y or G, X9=L, F, M or Y, the other Xn being any amino acid residue; L4: D[P/I/S]CX1X2CX3X4[H/R], wherein X2=A.S.V.G or T, X1, X3 and X4 are any amino acid residue and optionally comprising a motif LO: R[I/V/A]EG[H/D/A].—modifying said initial polynucleotide in order to substitute at least one of the residues X2 of motif L2 and Z or X4 of motif L3 and Z or X9 of motif L3 of said large subunit by a methionine.