Wu Guanghui, Corker Hazel, Orii Yutaka, Poole Robert K
Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK.
Arch Microbiol. 2004 Oct;182(2-3):193-203. doi: 10.1007/s00203-004-0699-8. Epub 2004 Aug 31.
Escherichia coli Hmp is a homologue of Ralstonia eutropha FHP, the first reported bacterial flavohaemoglobin, and functions in NO detoxification. Photolysis of CO-ligated Hmp in the presence of oxygen gave a photodissociable oxy species with k(on) 2.82x10(7) M(-1) s(-1) and k(off) 4.49x10(3) s(-1). The dissociation constant of the primary O(2) compound was 160 microM (25 degrees C, pH 7.0). In order to detect superoxide formation, ferric horseradish peroxidase was used. Hmp formed the oxy compound within milliseconds, followed by formation of compound III, arising from superoxide formation. The rate of superoxide formation was independent of oxygen concentration between 0.05 and 0.7 mM oxygen, suggesting a K(m) <0.05 mM. During prolonged oxidation of NADH, the spectral signals of Hmp decayed and iron was released in a process prevented by superoxide dismutase or catalase. NADH oxidation by purified Hmp was characterised by progressive slowing of oxygen uptake. Inclusion of NO, superoxide dismutase or catalase during NADH oxidation partially protected oxygen uptake, consistent with the formation, in the absence of NO, of reactive oxygen species that inhibit Hmp function. The results are discussed in relation to the tight control exerted on Hmp synthesis in vivo.
大肠杆菌Hmp是嗜麦芽窄食单胞菌FHP（首个被报道的细菌黄素血红蛋白）的同源物，具有一氧化氮解毒功能。在氧气存在的情况下，光解一氧化碳结合的Hmp会产生一种光可解离的氧合物种，其结合速率常数k(on)为2.82×10⁷ M⁻¹ s⁻¹，解离速率常数k(off)为4.49×10³ s⁻¹。主要氧合化合物的解离常数为160 μM（25℃，pH 7.0）。为了检测超氧化物的形成，使用了铁辣根过氧化物酶。Hmp在数毫秒内形成氧合化合物，随后形成由超氧化物形成导致的化合物III。超氧化物的形成速率在0.05至0.7 mM氧气浓度范围内与氧气浓度无关，表明其米氏常数K(m) <0.05 mM。在NADH的长时间氧化过程中，Hmp的光谱信号衰减，铁被释放，这一过程可被超氧化物歧化酶或过氧化氢酶阻止。纯化的Hmp对NADH的氧化表现为氧气摄取逐渐减慢。在NADH氧化过程中加入一氧化氮、超氧化物歧化酶或过氧化氢酶可部分保护氧气摄取，这与在无一氧化氮时形成抑制Hmp功能的活性氧物种一致。本文结合体内对Hmp合成的严格控制对结果进行了讨论。
