Gilles-Gonzalez M A, Gonzalez G, Perutz M F, Kiger L, Marden M C, Poyart C
Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.
Biochemistry. 1994 Jul 5;33(26):8067-73. doi: 10.1021/bi00192a011.
FixL's are chimeric heme protein kinases from symbiotic nitrogen-fixing Rhizobia. We have overexpressed three FixL variants in Escherichia coli. Bradyrhizobium japonicum FixL, a soluble dimeric protein, is the first full-length FixL to be purified. The other two proteins are soluble truncations of Rhizobium meliloti FixL, which is a membrane protein. One contains both heme and kinase domains and is dimeric; the other has only the heme domain and is monomeric. We find that all the FixL's bind oxygen and carbon monoxide non-cooperatively, with very low affinities due entirely to slow association rates. FixL P50's for oxygen are 17-76 mmHg. FixL's may sense nitric oxide and carbon monoxide in addition to oxygen, especially at the low oxygen pressures encountered in vivo. Autoxidation rates are about 50 times faster than that of sperm whale myoglobin. The carbon monoxide affinity of FixL's is about 300 times lower than that of myoglobin, resulting in the unusually low values of 7.5-17 for the partition constant, M = P50(O2)/P50(CO), between carbon monoxide and oxygen. Met-FixL's have their Soret absorption maximum at 395 nm instead of the typical 408 nm and a steep hydroxymet transition at pH > or = 9.3; these properties indicate a pentacoordinated high-spin ferric heme and suggest a sterically hindered hydrophobic heme pocket lacking a distal (E7) histidine. FixL is the first member of a new class of heme proteins, the heme-based sensors, distinct from the oxygen carriers and electron transporters. We expect that some of the novel properties of FixL will be characteristic of the class.
FixL是共生固氮根瘤菌中的嵌合血红素蛋白激酶。我们已在大肠杆菌中过表达了三种FixL变体。日本慢生根瘤菌FixL是一种可溶性二聚体蛋白，是首个被纯化的全长FixL。另外两种蛋白是苜蓿中华根瘤菌FixL的可溶性截短形式，苜蓿中华根瘤菌FixL是一种膜蛋白。一种同时包含血红素和激酶结构域，呈二聚体形式；另一种仅含有血红素结构域，呈单体形式。我们发现所有FixL均以非协同方式结合氧气和一氧化碳，亲和力极低，这完全是由于结合速率缓慢所致。FixL对氧气的P50值为17 - 76 mmHg。FixL除了能感知氧气外，可能还能感知一氧化氮和一氧化碳，尤其是在体内遇到的低氧压力条件下。自氧化速率比抹香鲸肌红蛋白快约50倍。FixL对一氧化碳的亲和力比肌红蛋白低约300倍，导致一氧化碳与氧气之间的分配常数M = P50(O2)/P50(CO)异常低，为7.5 - 17。高铁FixL的Soret吸收峰在395 nm而不是典型的408 nm，并且在pH≥9.3时具有陡峭的羟基高铁转变；这些特性表明存在一个五配位的高自旋铁血红素，并暗示存在一个空间位阻的疏水血红素口袋，缺乏远端（E7）组氨酸。FixL是一类新型血红素蛋白——基于血红素的传感器的首个成员，不同于氧载体和电子转运蛋白。我们预计FixL的一些新特性将是该类蛋白的特征。
