Lukat-Rodgers G S, Rodgers K R
Department of Chemistry, North Dakota State University, Fargo 58105, USA.
Biochemistry. 1997 Apr 8;36(14):4178-87. doi: 10.1021/bi9628230.
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.
本文报道了苜蓿根瘤菌FixL的两种可溶性截短形式（FixL和FixLN）亚铁形式的一氧化氮加合物的共振拉曼光谱。在室温下，亚铁FixL-NO和亚铁FixLN-NO均观察到四个同位素敏感振动。对于FixL*-NO，它们分别在558、525、450和1675 cm⁻¹处被观察到，并分别归属为六配位亚硝酰加合物的v(Fe-NO)、五配位亚硝酰加合物的v(Fe-NO)、六配位亚硝酰加合物的δ(Fe-NO)以及五配位亚硝酰加合物的v(N-O)。FixLN-NO同位素敏感带也观察到类似频率。基于v(Fe-NO)和δ(Fe-NO)模式的频率和光谱分离，得出Fe-N-O单元具有类似于亚铁血红蛋白和肌红蛋白亚硝酰加合物所观察到的弯曲几何结构。通过降低温度，这两种蛋白质均可转化为主要为五配位的亚硝酰加合物。在FixL*-NO和FixLN-NO的低温共振拉曼光谱中，558 cm⁻¹处的谱带强度显著降低，并且分别在529和526 cm⁻¹处观察到v(Fe-NO)5-c（五配位亚硝酰加合物的Fe-NO伸缩振动）。对这些亚硝酰加合物的v3和v8振动分析也支持在室温下FixL和FixLN同时存在五配位和六配位亚硝酰加合物，以及在低温下转化为主要为五配位亚硝酰加合物。这种温度依赖性的相互转化是可逆的。讨论了热可及五配位状态可能的生理相关性。FixLN-NO中v(Fe-NO)6-c半高宽比FixL-NO宽1.3倍，表明FixL*-NO中Fe-N-O几何结构更均匀。在FixLN-NO的低温光谱中，观察到第二个v(N-O)5-c谱带，表明在五配位FixLN-NO中可获得不止一种构象。五配位FixL*-NO未观察到第二个v(N-O)5-c，进一步表明FixL*中亚硝酰血红素的构象限制更大。涉及Fe-NO单元的振动变化表明激酶结构域影响血红素结构。根据导致激酶活性的配体依赖性介导的结构域间相互作用讨论了光谱差异。
