Characterization of ferrous FixL-nitric oxide adducts by resonance Raman spectroscopy.
Resonance Raman spectra of the nitric oxide adducts of the ferrous forms of two soluble truncations of Rhizobium meliloti FixL, FixL* and FixLN, are reported. At room temperature, four isotope sensitive vibrations are observed for both ferrous FixL*-NO and ferrous FixLN-NO. For FixL*-NO, they are observed at 558, 525, 450, and 1675 cm(-1) and are assigned to v(Fe-NO) of a six-coordinate nitrosyl adduct, v(Fe-NO) of a five-coordinate nitrosyl adduct, delta(Fe-NO) of a six-coordinate nitrosyl adduct, and v(N-O) of a five-coordinate nitrosyl adduct, respectively. Similar frequencies are observed for the FixLN-NO isotope sensitive bands. On the basis of the frequencies and spectral separation of the v(Fe-NO) and delta(Fe-NO) modes, the Fe-N-O unit is concluded to have a bent geometry similar to those observed for the nitrosyl adducts of ferrous hemoglobin and myoglobin. Both proteins can be converted to predominantly five-coordinate nitrosyl adducts by lowering the temperature. In low-temperature resonance Raman spectra of FixL*-NO and FixLN-NO, the 558 cm(-1) bands are significantly decreased in intensity and v(Fe-NO)5-c (the Fe-NO stretching vibration for the five-coordinate nitrosyl adduct) is observed at 529 and 526 cm(-1), respectively. Analysis of the v3 and v8 vibrations for these nitrosyl adducts also supports the presence of both five- and six-coordinate nitrosyl adducts of FixL* and FixLN at room temperature and the conversion to predominantly five-coordinate nitrosyl adducts at low temperatures. This temperature-dependent interconversion is reversible. The possible physiological relevance of the thermally accessible five-coordinate state is discussed. The width of v(Fe-NO)6-c at half-height is 1.3 times broader in FixLN-NO than in FixL*-NO, suggesting that the Fe-N-O geometry is more homogeneous in FixL*-NO. In low-temperature spectra of FixLN-NO, a second v(N-O)5-c band is observed, indicating that more than one conformation is attainable in the five-coordinate FixLN-NO. This second v(N-O)5-c is not observed for five-coordinate FixL*-NO, further suggesting a more conformationally restricted nitrosyl heme in FixL*. These variations in the vibrations involving the Fe-NO unit indicate that the kinase domain influences the heme structure. The spectral differences are discussed in terms of the interdomain interactions that result in ligation-dependent mediation of the kinase activity.
Lukat-Rodgers
G S
GS
Department of Chemistry, North Dakota State University, Fargo 58105, USA.
Rodgers
K R
KR
D001426:Bacterial Proteins / Q000737:chemistry*; D004227:Dithionite / Q000378:metabolism; D005296:Ferrous Compounds / Q000737:chemistry; D006420:Hemeproteins / Q000737:chemistry*; D000071677:Histidine Kinase; D009569:Nitric Oxide / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011494:Protein Kinases / Q000737:chemistry; D016962:Sinorhizobium meliloti / Q000737:chemistry*; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D013696:Temperature
D001426:Bacterial Proteins; D005296:Ferrous Compounds; D006420:Hemeproteins; D004227:Dithionite; D009569:Nitric Oxide; D011494:Protein Kinases; C067929:FixL protein, Bacteria; D000071677:Histidine Kinase