Lukat-Rodgers G S, Rexine J L, Rodgers K R
Department of Chemistry, North Dakota State University, Fargo 58105-5516, USA.
Biochemistry. 1998 Sep 29;37(39):13543-52. doi: 10.1021/bi981439v.
The pH-dependent behavior of the ferric forms of two soluble truncations of Rhizobium meliloti FixL, FixL (heme and kinase domains, functional), and FixLN (heme domain) are examined by UV-visible, resonance Raman, and electron paramagnetic resonance spectroscopy. Global analysis of UV-visible data indicates that the pKa for hydroxide binding is slightly higher in FixL than in FixLN. Spectroscopic data show that high-spin and low-spin hydroxide adducts of FixLN and FixL exist in a thermal spin-state equilibrium with a significant fraction of the heme in the high spin form at room temperature. FixLN and FixL differ from myoglobin and hemoglobin in that their hemes are not fully ligated by hydroxide ion under strongly alkaline conditions. In addition to the binding of hydroxide ion, both FixLN and FixL undergo additional alkaline transitions that involve the deprotonation of tyrosine residues. FixLN contains four tyrosine residues. One has a pKa of 9.6, which is indistinguishable from that for hydroxide binding to the heme. The other three tyrosines have pKas greater than 11. At pH 11, the alkaline species react with cyanide to yield the familiar low-spin cyanide adduct. Upon reduction of the heme iron, the alkaline forms of the FixL deletion derivatives are converted to their deoxy forms. Resonance Raman spectra reveal that the Fe-His stretching vibrations of deoxyFixLN and deoxyFixL are not measurably shifted from those of their neutral counterparts. Treatment of the alkaline deoxyFixLs with O2 yields the respective oxy forms. Spectroscopic evidence indicates that the loss of activity at elevated pH cannot be attributed solely to generation of a low-spin heme hydroxide. Involvement of one or more tyrosines in signal transmission between the heme and kinase domains of FixL is proposed.
通过紫外可见光谱、共振拉曼光谱和电子顺磁共振光谱研究了苜蓿根瘤菌FixL的两种可溶性截短形式（功能性的FixL（血红素和激酶结构域）和FixLN（血红素结构域））的铁离子形式的pH依赖性行为。紫外可见数据的全局分析表明，FixL中氢氧化物结合的pKa略高于FixLN。光谱数据表明，FixLN和FixL的高自旋和低自旋氢氧化物加合物在热自旋态平衡中存在，室温下血红素的很大一部分处于高自旋形式。FixLN和FixL与肌红蛋白和血红蛋白的不同之处在于，在强碱性条件下，它们的血红素没有被氢氧根离子完全配位。除了氢氧根离子的结合外，FixLN和FixL都经历了涉及酪氨酸残基去质子化的额外碱性转变。FixLN含有四个酪氨酸残基。其中一个的pKa为9.6，与氢氧根与血红素结合的pKa无法区分。其他三个酪氨酸的pKa大于11。在pH 11时，碱性物质与氰化物反应生成常见的低自旋氰化物加合物。血红素铁还原后，FixL缺失衍生物的碱性形式转化为脱氧形式。共振拉曼光谱显示，脱氧FixLN和脱氧FixL的Fe-His伸缩振动与其中性对应物相比没有明显位移。用O2处理碱性脱氧FixL会产生相应的氧合形式。光谱证据表明，在较高pH下活性的丧失不能仅仅归因于低自旋血红素氢氧化物的生成。有人提出一个或多个酪氨酸参与了FixL血红素和激酶结构域之间的信号传递。
