Leu65 in the heme distal side is critical for the stability of the Fe(II)-O2 complex of YddV, a globin-coupled oxygen sensor diguanylate cyclase.

YddV is a globin-coupled oxygen sensor enzyme in that O(2) binding to the Fe(II) heme in the sensor domain substantially enhances its diguanylate cyclase activity. The Fe(III) heme-bound enzyme is also the active form. Amino acid sequence comparisons indicate that Leu65 is well conserved in globin-coupled oxygen sensor enzymes. Absorption spectra of the Fe(III) heme complexes of L65G, L65M, L65Q and L65T mutants of the isolated heme domain of YddV (YddV-heme) were substantially different from that of the wild-type protein. Specifically, Soret bands of the 6-coordinated high-spin Fe(III) complexes of mutant proteins (with H(2)O and His98 as axial ligands) were located at around 403-406 nm, distinct from that (391 nm) of the 5-coordinated high-spin Fe(III) complex of wild-type protein with His98 as the axial ligand. The autooxidation rate constant (>0.10 min(-1)) of the Fe(II)-O(2) complex of L65G was substantially higher than that (0.011 min(-1)) of the wild-type protein. Affinities of O(2) for the Fe(II) complexes of L65G and L65T were markedly higher than that for the wild-type protein. Thus, we suggest that the well-conserved Leu65 located in the heme distal side is critical for restricting water access to the heme distal side to avoid rapid autooxidation of YddV, which needs a stable Fe(II)-O(2) complex with a low autooxidation rate.