Nitrosyl adducts of FixL as probes of heme environment.
This report presents a spectroscopic investigation of the nitrosyl adducts of FixL, the sensor in the signal transduction system responsible for regulating nitrogen fixation in Rhizobium meliloti. Variable-temperature resonance Raman (RR), electron spin resonance (ESR), and variable-temperature UV-visible absorption data are presented for the ferrous NO adducts of two FixL deletion derivatives, FixLN (the heme-containing domain) and FixL* (a functional heme-kinase). A temperature-dependent equilibrium is observed between the five-coordinate (5-c) and six-coordinate (6-c) ferrous nitrosyl adducts, with lower temperatures favoring formation of the 6-c nitrosyl adduct. This equilibrium is perturbed as the solution freezes, and the amount of 5-c FixL-NO increases sharply until a nearly constant ratio of 6-c to 5-c adducts is obtained. Complexation between the heme domain of FixL and its response regulator, FixJ, is revealed through specfic FixJ-induced increase in the energy separation between 5-c and 6-c FixL-NO. Ferric nitrosyl adducts of FixL* and FixLN autoreduce to their corresponding ferrous nitrosyl adducts. The kinetic behavior of this reduction is monophasic for FixL*-NO, while the reaction for ferric FixLN-NO is biphasic. These results suggest conformational inhomogeneity in the heme pocket of FixLN and conformational homogeneity in that of FixL*. Hence the kinase domain plays a role in distal pocket conformational stability. Implications for the signal transduction mechanism are discussed.
Rodgers
K R
KR
Department of Chemistry, North Dakota State University, Fargo 58105, USA. rodgers@plains.nodak.edu
Lukat-Rodgers
G S
GS
Tang
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L
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D004227:Dithionite / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000378:metabolism*; D006420:Hemeproteins / Q000737:chemistry* / Q000378:metabolism*; D000071677:Histidine Kinase; D007700:Kinetics; D008958:Models, Molecular; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism; D009603:Nitroso Compounds / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D016962:Sinorhizobium meliloti; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D013696:Temperature; D013816:Thermodynamics
D001426:Bacterial Proteins; D006420:Hemeproteins; D009603:Nitroso Compounds; D004227:Dithionite; D009569:Nitric Oxide; D006418:Heme; C067929:FixL protein, Bacteria; D000071677:Histidine Kinase; D010100:Oxygen