Fang T Y, Simplaceanu V, Tsai C H, Ho N T, Ho C
Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, USA.
Biochemistry. 2000 Nov 14;39(45):13708-18. doi: 10.1021/bi001115i.
Site-directed mutagenesis has been used to construct three recombinant mutant hemoglobins (rHbs), rHb(beta L105W), rHb(alpha D94A/betaL105W), and rHb(alpha D94A). rHb(beta L105W) is designed to form a new hydrogen bond from beta 105Trp to alpha 94Asp in the alpha(1)beta(2) subunit interface to lower the oxygen binding affinity by stabilizing the deoxy quaternary structure. We have found that rHb(beta L105W) does indeed possess a very low oxygen affinity and maintains normal cooperativity (P(50) = 28.2 mmHg, n(max) = 2.6 in 0.1 M sodium phosphate at pH 7.4) compared to those of Hb A (P(50) = 9.9 mmHg, n(max) = 3.2 at pH 7.4). rHb(alpha D94A/beta L105W) and rHb(alpha D94A) are expressed to provide evidence that rHb(betaL 105W) does form a new H-bond from beta 105Trp to alpha 94Asp in the alpha(1)beta(2) subunit interface of the deoxy quaternary structure. Our multinuclear, multidimensional nuclear magnetic resonance (NMR) studies on (15)N-labeled rHb(beta L105W) have identified the indole nitrogen-attached (1)H resonance of beta 105Trp for rHb(beta L105W). (1)H NMR studies on Hb A and mutant rHbs have been used to investigate the structural basis for the low O(2) affinity of rHb(beta L105W). Our NMR results provide evidence that rHb(beta L105W) forms a new H-bond from beta 105Trp to alpha 94Asp in the alpha(1)beta(2) subunit interface of the deoxy quaternary structure. The NMR results also show that these three rHbs can switch from the R quaternary structure to the T quaternary structure in their ligated state upon addition of an allosteric effector, inositol hexaphosphate. We propose that the low O(2) affinity of rHb(beta L105W) is due to the formation of a new H-bond between alpha 105Trp and alpha 94Asp in the deoxy quaternary structure.
定点诱变已被用于构建三种重组突变血红蛋白（rHb），即rHb（βL105W）、rHb（αD94A/βL105W）和rHb（αD94A）。rHb（βL105W）的设计目的是在α(1)β(2)亚基界面形成一个从β105色氨酸到α94天冬氨酸的新氢键，通过稳定脱氧四级结构来降低氧结合亲和力。我们发现，与Hb A（pH 7.4时P(50)=9.9 mmHg，n(max)=3.2）相比，rHb（βL105W）确实具有非常低的氧亲和力并保持正常的协同性（pH 7.4时在0.1 M磷酸钠中P(50)=28.2 mmHg，n(max)=2.6）。表达rHb（αD94A/βL105W）和rHb（αD94A）是为了提供证据，证明rHb（βL105W）在脱氧四级结构的α(1)β(2)亚基界面确实形成了一个从β105色氨酸到α94天冬氨酸的新氢键。我们对（15）N标记的rHb（βL105W）进行的多核、多维核磁共振（NMR）研究确定了rHb（βL105W）中β105色氨酸的吲哚氮连接的（1）H共振。对Hb A和突变rHb的（1）H NMR研究已被用于研究rHb（βL105W）低O(2)亲和力的结构基础。我们的NMR结果提供了证据，证明rHb（βL105W）在脱氧四级结构的α(1)β(2)亚基界面形成了一个从β105色氨酸到α94天冬氨酸的新氢键。NMR结果还表明，在添加变构效应剂肌醇六磷酸后，这三种rHb在其结合状态下可以从R四级结构转变为T四级结构。我们提出，rHb（βL105W）的低O(2)亲和力是由于在脱氧四级结构中α105色氨酸和α94天冬氨酸之间形成了一个新氢键。
