Sivakolundu S G, Mabrouk P A
Department of Chemistry, Northeastern University, Boston, Massachusetts 02115, USA.
Protein Sci. 2001 Nov;10(11):2291-300. doi: 10.1110/ps.15601.
Recently, we found that ferricytochrome c (ferricyt c) undergoes significant structural changes in mixed aqueous-nonaqueous media, resulting in the formation of a mixture of alkaline-like species. The equilibrium composition of this mixture of species is dependent on the dielectric constant of the mixed solvent medium. One-dimensional (1D) and two-dimensional (2D) (1)H nuclear magnetic resonance (NMR) methods have now been used to study these alkaline-like forms in 30% acetonitrile-water solution. A native-like (M80-ligated) III* form, two lysine-ligated forms (IVa* and IVb*), and a hydroxide-ligated form (V*) were observed. Heme proton resonance assignments for these forms were accomplished using 1D (1)H NMR and 2D nuclear Overhauser effect spectroscopy methods at 20 degrees C and 35 degrees C. The chemical exchange between the alkaline forms in 30% acetonitrile solution facilitated heme proton resonance assignments. Based on examination of the heme proton chemical shifts and several highly conserved amino acid residues, the electronic structure, secondary structure, and hydrogen bond network in the vicinity of the heme in the III* form were found to be intact. Similarly, the heme electronic structure of the IVa* form was found to be comparable to that of the IVa form. Differences in the order of the heme methyl resonances in the IVb* form, however, suggest that the heme active site in this form is somewhat different from that observed in aqueous alkaline solution. In addition, resonance assignments for the 8- and 3-methyl heme protons were made for the hydroxide-ligated V* form for the first time. The observation of chemical exchange peaks between all species except IVb* and IVa* or V* was used to propose an exchange pathway between the different forms of ferricyt c in 30% acetonitrile solution. This pathway may be biologically significant because ferricyt c, which resides in the intermembrane space of mitochondria, is exposed to medium of relatively low dielectric constant when it interacts with the mitochondrial membrane.
最近，我们发现高铁细胞色素c（ferricyt c）在混合水-非水介质中会发生显著的结构变化，从而形成类似碱性物种的混合物。该物种混合物的平衡组成取决于混合溶剂介质的介电常数。现在已使用一维（1D）和二维（2D）氢核磁共振（NMR）方法来研究30%乙腈-水溶液中的这些类似碱性的形式。观察到一种类似天然的（M80连接）III形式、两种赖氨酸连接形式（IVa和IVb*）以及一种氢氧化物连接形式（V*）。在20℃和35℃下，使用1D氢核磁共振和二维核Overhauser效应光谱方法完成了这些形式的血红素质子共振归属。30%乙腈溶液中碱性形式之间的化学交换促进了血红素质子共振归属。基于对血红素质子化学位移和几个高度保守的氨基酸残基的研究，发现III形式中血红素附近的电子结构、二级结构和氢键网络是完整的。同样，发现IVa形式的血红素电子结构与IVa形式相当。然而，IVb形式中血红素甲基共振顺序的差异表明，这种形式中的血红素活性位点与在碱性水溶液中观察到的有所不同。此外，首次对氢氧化物连接的V形式的8-甲基和3-甲基血红素质子进行了共振归属。观察到除IVb与IVa或V*之外的所有物种之间的化学交换峰，据此提出了30%乙腈溶液中不同形式的ferricyt c之间的交换途径。这条途径可能具有生物学意义，因为存在于线粒体内膜间隙的ferricyt c在与线粒体膜相互作用时会暴露于相对低介电常数的介质中。
