Important Roles of Tyr43 at the Putative Heme Distal Side in the Oxygen Recognition and Stability of the Fe(II)-O2 Complex of YddV, a Globin-Coupled Heme-Based Oxygen Sensor Diguanylate Cyclase

YddV from Escherichia coli (Ec) is a novel globin-coupled heme-based oxygen sensor protein displaying diguanylate cyclase activity in response to oxygen availability. In this study, we quantified the turnover numbers of the active [Fe(III), 0.066 min(-1); Fe(II)-O-2 and Fe(II)-CO, 0.022 min(-1)] [Fe(III), Fe(III)-protoporphyrin IX complex; Fe(II), Fe(II)-protoporphyrin IX complex] and inactive forms [Fe(II) and Fe(II)-NO, <0.01 min(-1)] of YddV for the first time. Our data indicate that the YddV reaction is the rate-determining step for two consecutive reactions coupled with phosphodiesterase Ec DOS activity on cyclic di-GMP (c-di-GMP) [turnover number of Ec DOS-Fe(II)-O-2, 61 min(-1)]. Thus, O-2 binding and the heme redox switch of YddV appear to be critical factors in the regulation of c-di-GMP homeostasis. The redox potential and autoxidation rate of heme of the isolated heme domain of YddV (YddV-heme) were determined to be -17 mV versus the standard hydrogen electrode and 0.0076 min(-1), respectively. The Fe(II) complexes of Y43A and Y43L mutant proteins (residues at the heme distal side of the isolated heme-bound globin domain of YddV) exhibited very low O-2 affinities, and thus, their Fe(II)-O-2 complexes were not detected on the spectra. The O-2 dissociation rate constant of the Y43W protein was > 150 s(-1), which is significantly larger than that of the wildtype protein (22 s(-1)). The autoxidation rate constants of the Y43F and Y43W mutant proteins were 0.069 and 0.12 min(-1), respectively, which are also markedly higher than that of the wild-type protein. The resonance Raman frequencies representing nu(Fe)_(O2) (559 cm(-1)) of the Fe(II)-O-2 complex and nu(Fe-CO) (505 cm(-1)) of the Fe(II)-CO complex of Y43F differed from those (nu(Fe-O2), 565 cm(-1); nu(Fe-CO), 495 cm(-1)) of the wild-type protein, suggesting that Tyr43 forms hydrogen bonds with both O-2 and CO molecules. On the basis of the results, we suggest that Tyr43 located at the heme distal side is important for the O-2 recognition and stability of the Fe(II)-O-2 complex, because the hydroxyl group of the residue appears to interact electrostatically with the O-2 molecule bound to the Fe(II) complex in YddV. Our findings clearly support a role of Tyr in oxygen sensing, and thus modulation of overall conversion from GTP to pGpG via c-di-GMP catalyzed by YddV and Ec DOS, which may be applicable to other globin-coupled oxygen sensor enzymes.