Sensory mechanism of oxygen sensor FixL from Rhizobium meliloti:: Crystallographic, mutagenesis and resonance Raman spectroscopic studies

FixL of Rhizobium meliloti (RmFixL) is a sensor histidine kinase of the two-component system, which regulates the expression of the genes related to nitrogen fixation in the root nodule in response to the O-2 levels. The crystal structure of the sensor domain of FixL (RmFixLH), which contains a heme (Fe-porphyrin) as a sensing site, was determined at 1.4 Angstrom resolution. Based on the structural and spectroscopic analyses, we propose the O-2 sensing mechanism that differs from the case proposed in BjFixLH as follows; conformational changes in the F/G loop, which are induced by steric repulsion between the bent-bound O-2 and the Ile209 side-chain, would be transmitted to the histidine kinase domain. Interaction between the iron-bound O-2, and Ile209 was also observed in the resonance Raman spectra of RmFixLH as evidenced by the fact that the Fe-O-2 and Fe-CN stretching frequencies were shifted from 575 to 570 cm(-1) (Fe-O-2), and 504 to 499 cm(-1), respectively, as the result of the replacement of Ile209 with an Ala residue. In the I209A mutant of RmFixL, the O-2 sensing activity was destroyed, thus confirming our proposed mechanism. (C) 2000 Academic Press.