Characterization of Ferrous FixL−Nitric Oxide Adducts by Resonance Raman Spectroscopy

Abstract

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 ν(Fe−NO) of a six-coordinate nitrosyl adduct, ν(Fe−NO) of a five-coordinate nitrosyl adduct, δ(Fe−NO) of a six-coordinate nitrosyl adduct, and ν(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 ν(Fe−NO) and δ(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 ν(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 ν₃ and ν₈ 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 ν(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 ν(N−O)_5-c band is observed, indicating that more than one conformation is attainable in the five-coordinate FixLN−NO. This second ν(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.