Pesce Alessandra, Nardini Marco, Ascenzi Paolo, Geuens Eva, Dewilde Sylvia, Moens Luc, Bolognesi Martino, Riggs Austen F, Hale Angela, Deng Pengchi, Nienhaus G Ulrich, Olson John S, Nienhaus Karin
Department of Physics-INFM and Center for Excellence in Biomedical Research, University of Genova, Via Dodecaneso 33, 16146 Genova, Italy.
J Biol Chem. 2004 Aug 6;279(32):33662-72. doi: 10.1074/jbc.M403597200. Epub 2004 May 25.
The mini-hemoglobin from Cerebratulus lacteus (CerHb) belongs to a class of globins containing the polar Tyr-B10/Gln-E7 amino acid pair that normally causes low rates of O2 dissociation and ultra-high O2 affinity, which suggest O2 sensing or NO scavenging functions. CerHb, however, has high rates of O2 dissociation (kO2 = 200-600 s(-1)) and moderate O2 affinity (KO2) approximately 1 microm(-1)) as a result of a third polar amino acid in its active site, Thr-E11. When Thr-E11 is replaced by Val, kO2 decreases 1000-fold and KO2 increases 130-fold at pH 7.0, 20 degrees C. The mutation also shifts the stretching frequencies of both heme-bound and photodissociated CO, indicating marked changes of the electrostatic field at the active site. The crystal structure of Thr-E11 --> Val CerHbO2 at 1.70 A resolution is almost identical to that of the wild-type protein (root mean square deviation of 0.12 A). The dramatic functional and spectral effects of the Thr-E11 --> Val mutation are due exclusively to changes in the hydrogen bonding network in the active site. Replacing Thr-E11 with Val "frees" the Tyr-B10 hydroxyl group to rotate toward and donate a strong hydrogen bond to the heme-bound ligand, causing a selective increase in O2 affinity, a decrease of the rate coefficient for O2 dissociation, a 40 cm(-1) decrease in nuCO of heme-bound CO, and an increase in ligand migration toward more remote intermediate sites.
来自乳光链蛇（CerHb）的微型血红蛋白属于一类球蛋白，其含有极性的Tyr-B10/Gln-E7氨基酸对，通常导致低氧解离速率和超高氧亲和力，这表明具有氧传感或一氧化氮清除功能。然而，由于其活性位点中的第三个极性氨基酸Thr-E11，CerHb具有高氧解离速率（kO2 = 200 - 600 s(-1)）和中等氧亲和力（KO2约为1 μmol(-1)）。当Thr-E11被Val取代时，在pH 7.0、20℃条件下，kO2降低1000倍，KO2增加130倍。该突变还改变了血红素结合的和光解离的CO的伸缩频率，表明活性位点处的静电场发生了显著变化。Thr-E11→Val CerHbO2在1.70 Å分辨率下的晶体结构与野生型蛋白几乎相同（均方根偏差为0.12 Å）。Thr-E11→Val突变产生的显著功能和光谱效应完全归因于活性位点氢键网络的变化。用Val取代Thr-E11使Tyr-B10羟基“自由”旋转并向血红素结合的配体提供强氢键，导致氧亲和力选择性增加、氧解离速率系数降低、血红素结合的CO的νCO降低40 cm(-1)，以及配体向更远的中间位点迁移增加。
