Spectroscopic and kinetic studies of FixL, the heme-based oxygen-sensing protein from Sinorhizobium meliloti: Implications for the mechanism of signal transduction.

摘要

The O2-sensor protein SmFixL is the sensor-kinase of a two-component system that regulates the transcription of nif and fix genes in Sinorhizobium meliloti. It comprises a heme-containing PAS domain and a histidine kinase domain. Binding and release of O2 by its heme modulate the kinase activity, which is required for transcriptional activation of its response regulator, SmFixJ. In this dissertation, three projects designed to further elucidate the inter-domain communication between the heme and kinase are presented.; First, the reductive nitrosylation of ferric SmFixL is discussed. Biphasic kinetics, as monitored by visible spectroscopy, were observed for the reductive nitrosylation of ferric SmFixLN (heme domain). The reaction for SmFixL* (functional heme-kinase) is monophasic. This suggests conformational homogeneity in the heme pocket of SmFixL*, which is consistent with previous evidence for conformational constraints in the distal heme pocket imposed by the kinase domain.; Second, characterization of a conformational intermediate generated upon photolysis of ferrous FixL-CO is presented. The photolabile nature of the Fe-CO bond in heme proteins allows efficient laser-induced dissociation of ferrous SmFixL-CO complexes. This provides a trigger for initiating conversion of kinase-inhibited SmFixL* (CO-bound) to kinase-active deoxySmFixL*. Using time-resolved resonance Raman spectroscopy, compression of the Fe-His bond after the photolysis of the SmFixL-CO adducts is evidenced by an upshifted nu(Fe-His) at 218 cm-1. This compression occurs before the protein reaches the equilibrium deoxy state, in which the kinase is active. This observation indicates that the proximal heme pocket is involved in the inter-domain signaling between heme and kinase.; Finally, a kinetic study of CO recombination to ferrous SmFixL* is presented. This study probes the intra-molecular conformational changes that are coupled to the binding and release of heme ligands. Using time-resolved absorbance spectroscopy, a single kinetic phase is observed for CO recombination to SmFixLN. However, SmFixL* rebinds CO via two phases, which are attributed to ligation-coupled interconversion of two SMFixL* conformers. Relevance of these conformers to signal transduction is discussed.; Based on these results and previous structural and spectroscopic characterization of SmFixL, a sequence of signal transduction events in SmFixL is proposed.