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 Diguanyla

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• 作者：Kenichi Kitanishi ; Kazuo Kobayashi ; Yuriko Kawamura ; Izumi Ishigami ; Takashi Ogura ; Kyosuke Nakajima ; Jotaro Igarashi ; Atsunari Tanaka ; Toru Shimizu
• 刊名：Biochemistry
• 出版年：2010
• 出版时间：December 14, 2010
• 年：2010
• 卷：49
• 期：49
• 页码：10381-10393
• 全文大小：1106K
• 年卷期：v.49,no.49(December 14, 2010)
• ISSN：1520-4995

文摘

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)−O₂ 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)−O₂, 61 min⁻¹]. Thus, O₂ 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 O₂ affinities, and thus, their Fe(II)−O₂ complexes were not detected on the spectra. The O₂ 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−O2 (559 cm⁻¹) of the Fe(II)−O₂ complex and ν_Fe−CO (505 cm⁻¹) of the Fe(II)−CO complex of Y43F differed from those (ν_Fe−O2, 565 cm⁻¹; ν_Fe−CO, 495 cm⁻¹) of the wild-type protein, suggesting that Tyr43 forms hydrogen bonds with both O₂ and CO molecules. On the basis of the results, we suggest that Tyr43 located at the heme distal side is important for the O₂ recognition and stability of the Fe(II)−O₂ complex, because the hydroxyl group of the residue appears to interact electrostatically with the O₂ 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.