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

Abstract

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 wild-type 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 ν(Fe-O(2)) (559 cm(-1)) of the Fe(II)-O(2) complex and ν(Fe-CO) (505 cm(-1)) of the Fe(II)-CO complex of Y43F differed from those (ν(Fe-O(2)), 565 cm(-1); ν(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.

Heme-based Globin-coupled Oxygen Sensors: Linking Oxygen Binding to Functional Regulation of Diguanylate Cyclase, Histidine Kinase, and Methyl-accepting Chemotaxis

“…CO binding to the Fe(II) heme complex also enhanced catalysis. These gas-induced catalytic enhancements and the redox-dependent catalytic regulation of AfGcHK are similar to those observed for YddV (32).…”

“…Contrary to expectations, heme-free forms and heme-binding domain-truncated forms of YddV, AfGcHK, and EcDOS have sufficient catalytic activity (32,44,72,94). Instead of stimulating catalysis, the heme iron complex on the heme-based oxygen sensor serves to suppress catalysis, and oxygen association/dissociation releases this catalytic suppression.…”

“…3), and physicochemical properties of mutant proteins. Again, the hydroxyl of Tyr-45(B10) interacts with O 2 in the heme Fe(II)-O 2 complex and stabilizes the complex via hydrogen bond donation, as observed for YddV (32).…”

“…c-diGMP is an important bacterial second messenger involved in mobility, virulence, development, cell-cell communication, and biofilm formation (32)(33)(34). In E. coli, DGC-mediated synthesis of c-diGMP is carried out by YddV (or E. coli DosC (EcDosC)), one of the GCSs, whereas degradation of c-diGMP is mediated by the PDE activity of another hemebased oxygen sensor, E. coli DOS (EcDOS (direct oxygen sensor); or EcDosP) (Figs.…”

“…Full-length Enzymes Are Dimers or Tetramers-The DGCGCSs identified and characterized to date, such as HemAT-Bs (22), AvGReg (35), BpeGReg (36), HemDGC (37), and YddV (32,38,39), are dimers or tetramers. Such an arrangement suggests that each subunit catalyzes the cyclization reaction possibly in synergy with other subunits in the dimer/tetramer.…”