Wu Y, Basti M, Gambacurta A, Chiancone E, Ascoli F, La Mar G N
Department of Chemistry, University of California, Davis 95616, USA.
Biochim Biophys Acta. 1996 Dec 5;1298(2):261-75. doi: 10.1016/s0167-4838(96)00137-9.
The active-site structure of the paramagnetic cyanomet complex of the cooperative homodimeric hemoglobin from Scapharca inaequivalvis has been investigated by solution homonuclear NMR. In spite of the large size (32 kDa), the residues on the key proximal F- and distal E-helices could be sequence-specifically assigned and placed in the heme pocket in a manner common to diamagnetic systems. These backbone assignments were greatly facilitated by the significant dispersion of backbone chemical shifts by the highly anisotropic paramagnetic susceptibility tensor of the low-spin ferric state. The remainder of the residues in contact with the heme are assigned based on unique contacts to the heme predicted by the crystal structure and the observations of scalar connectivities diagnostic for the residues. The magnitude of the dipolar shifts for non-ligated residues was used to determine the anisotropy and orientation of the paramagnetic susceptibility tensor, and the major axis found tilted from the normal in a manner similar to that found for the Fe-CO unit in the crystal structure. The combination of NOESY inter-residue and heme-residue contacts, paramagnetic-induced relaxation and correlation between observed and dipolar shifts provide a description of the heme cavity in cyanomet Hb that is essentially the same as found in the carbonmonoxy Hb crystal structure. The pattern of both the heme methyl dominant contact shifts and the heme meso-proton dominant dipolar shifts are shown to be consistent with the orientation of the axial His. It is concluded that the present homonuclear NMR methods allow effective solution structure determination in the cyanomet form for dimeric Hb and suggest profitable extension to the tetrameric vertebrate hemoglobins.
通过溶液同核核磁共振研究了不等边毛蚶协同同二聚体血红蛋白的顺磁氰化物配合物的活性位点结构。尽管其分子量较大（32 kDa），但关键的近端F螺旋和远端E螺旋上的残基能够进行序列特异性归属，并以与抗磁体系相同的方式置于血红素口袋中。低自旋铁态的高度各向异性顺磁磁化率张量使主链化学位移有显著分散，极大地促进了这些主链归属。基于晶体结构预测的与血红素的独特接触以及对残基具有诊断性的标量连接性观察结果，对与血红素接触的其余残基进行了归属。未配位残基的偶极位移大小用于确定顺磁磁化率张量的各向异性和取向，发现其主轴相对于法线倾斜，方式与晶体结构中Fe-CO单元的情况相似。NOESY残基间和血红素-残基接触、顺磁诱导弛豫以及观察到的位移与偶极位移之间的相关性相结合，提供了对氰化高铁血红蛋白血红素腔的描述，该描述与一氧化碳血红蛋白晶体结构中的情况基本相同。血红素甲基主导的接触位移和血红素中质子主导的偶极位移模式均显示与轴向组氨酸的取向一致。得出的结论是，目前的同核核磁共振方法能够有效地确定二聚体血红蛋白氰化高铁形式的溶液结构，并表明对四聚体脊椎动物血红蛋白进行有益的扩展是可行的。
