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 O2 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 H2O 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)–O2 complex of L65G was substantially higher than that (0.011 min− 1) of the wild-type protein. Affinities of O2 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)–O2 complex with a low autooxidation rate.

YddV is a heme-based globin-coupled oxygen sensor diguanylate cyclase. Leu65 is critical to block the entry of water molecules into the heme iron complex and maintain an adequate autooxidation rate. Leu65 may be critical for regulating the function of the oxygen sensor enzyme.Figure optionsDownload as PowerPoint slide