HEME-BASED SENSORS, EXEMPLIFIED BY THE KINASE FIXL, ARE A NEW CLASS OF HEME PROTEIN WITH DISTINCTIVE LIGAND-BINDING AND AUTOXIDATION

FixL's are chimeric heme protein kinases from symbiotic nitrogen-fixin g Rhizobia. We have overexpressed three FixL variants in Escherichia c oli. Bradyrhizobium japonicum FixL, a soluble dimeric protein, is the first full-length FixL to be purified. The other two proteins are solu ble truncations of Rhizobium meliloti FixL, which is a membrane protei n. 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 aff inities due entirely to slow association rates. FixL P-50'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 sp erm whale myoglobin. The carbon monoxide affinity of FixL's is about 3 00 times lower than that of myoglobin, resulting in the unusually low values of 7.5-17 for the partition constant, M = P-50(O-2)/P-50(CO), b etween carbon monoxide and oxygen. Met-FixL's have their Soret absorpt ion maximum at 395 nm instead of the typical 408 nm and a steep hydrox ymet transition at pH greater than or equal to 9.3; these properties i ndicate a pentacoordinated high-spin ferric heme and suggest a sterica lly hindered hydrophobic heme pocket lacking a distal (E7) histidine. FixL is the first member of a new class of heme proteins, the heme-bas ed sensors, distinct from the oxygen carriers and electron transporter s. We expect that some of the novel properties of FixL will be charact eristic of the class.