Brunori M, Cutruzzolà F, Savino C, Travaglini-Allocatelli C, Vallone B, Gibson Q H
Department of Biochemical Sciences and CNR Center of Molecular Biology, University of Rome "La Sapienza," Rome, Italy.
Biophys J. 1999 Mar;76(3):1259-69. doi: 10.1016/S0006-3495(99)77289-9.
A triple mutant of sperm whale myoglobin (Mb) [Leu(B10) --> Tyr, His(E7) --> Gln, and Thr(E10) --> Arg, called Mb-YQR], investigated by stopped-flow, laser photolysis, crystallography, and molecular dynamics (MD) simulations, proved to be quite unusual. Rebinding of photodissociated NO, O2, and CO from within the protein (in a "geminate" mode) allows us to reach general conclusions about dynamics and cavities in proteins. The 3D structure of oxy Mb-YQR shows that bound O2 makes two H-bonds with Tyr(B10)29 and Gln(E7)64; on deoxygenation, these two residues move toward the space occupied by O2. The bimolecular rate constant for NO binding is the same as for wild-type, but those for CO and O2 binding are reduced 10-fold. While there is no geminate recombination with O2 and CO, geminate rebinding of NO displays an unusually large and very slow component, which is pretty much abolished in the presence of xenon. These results and MD simulations suggest that the ligand migrates in the protein matrix to a major "secondary site," located beneath Tyr(B10)29 and accessible via the motion of Ile(G8)107; this site is different from the "primary site" identified by others who investigated the photolyzed state of wild-type Mb by crystallography. Our hypothesis may rationalize the O2 binding properties of Mb-YQR, and more generally to propose a mechanism of control of ligand binding and dissociation in hemeproteins based on the dynamics of side chains that may (or may not) allow access to and direct temporary sequestration of the dissociated ligand in a docking site within the protein. This interpretation suggests that very fast (picosecond) fluctuations of amino acid side chains may play a crucial role in controlling O2 delivery to tissue at a rate compatible with physiology.
通过停流法、激光光解、晶体学和分子动力学（MD）模拟研究的抹香鲸肌红蛋白（Mb）的三重突变体[Leu(B10)→Tyr、His(E7)→Gln和Thr(E10)→Arg，称为Mb - YQR]被证明非常特别。光解离的NO、O2和CO从蛋白质内部重新结合（以“双分子”模式）使我们能够得出关于蛋白质动力学和空腔的一般结论。氧合Mb - YQR的三维结构表明，结合的O2与Tyr(B10)29和Gln(E7)64形成两个氢键；脱氧时，这两个残基向O2占据的空间移动。NO结合的双分子速率常数与野生型相同，但CO和O2结合的速率常数降低了10倍。虽然不存在与O2和CO的双分子复合，但NO的双分子重新结合显示出异常大且非常缓慢的成分，在氙气存在下几乎完全消失。这些结果和MD模拟表明，配体在蛋白质基质中迁移到一个主要的“二级位点”，该位点位于Tyr(B10)29下方，可通过Ile(G8)107的运动到达；这个位点与其他通过晶体学研究野生型Mb光解状态的人所确定的“一级位点”不同。我们的假设可以解释Mb - YQR的O2结合特性，更普遍地提出一种基于侧链动力学控制血红素蛋白中配体结合和解离的机制，这种动力学可能（或可能不）允许解离的配体进入并直接暂时隔离在蛋白质内的对接位点。这种解释表明，氨基酸侧链的非常快速（皮秒）的波动可能在以与生理学兼容的速率控制O2向组织的输送中起关键作用。
