Shimoni E, Tsfadia Y, Nachliel E, Gutman M
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel.
Biophys J. 1993 Feb;64(2):472-9. doi: 10.1016/S0006-3495(93)81389-4.
Time resolved fluorimetry was employed to monitor the geminate recombination between proton and excited pyranine anion locked, together with less than 30 water molecules, inside the heme binding site of Apomyoglobin (sperm whale). The results were analyzed by a numerical reconstruction of the differential rate equation for time-dependent diffusion controlled reaction with radiating boundaries using N. Agmon's procedure (Huppert, Pines, and Agmon, 1990, J. Opt. Soc. Am. B., 7:1541-1550). The analysis of the curve provided the effective dielectric constant of the proton permeable space in the cavity and the diffusion coefficient of the proton. The electrostatic potential within the cavity was investigated by the equations given by Gilson et al. (1985, J. Mol. Biol., 183:503-516). According to this analysis the dielectric constant of the protein surrounding the site is epsilon prot < or = 6.5. The diffusion coefficient of the proton in the heme binding site of Apomyoglobin-pyranine complex is D = 4 x 10(-5) cm2/s. This value is approximately 50% of the diffusion coefficient of proton in water. The lower value indicates enhanced ordering of water in the cavity, a finding which is corroborated by a large negative enthropy of binding delta S0 = -46.6 cal.mole-1 deg-1. The capacity of a small cavity in a protein to retain a proton had been investigated through the mathematical reconstruction of the dynamics. It has been demonstrated that Coulombic attraction, as large as delta psi of energy coupling membrane, is insufficient to delay a free proton for a time frame comparable to the turnover time of protogenic sites.
采用时间分辨荧光法监测质子与激发态吡喃阴离子之间的双分子复合，它们被锁定在精子鲸肌红蛋白血红素结合位点内，周围水分子少于30个。使用N. Agmon的方法（Huppert、Pines和Agmon，1990年，《美国光学学会杂志B》，7:1541 - 1550），通过对具有辐射边界的时间相关扩散控制反应的微分速率方程进行数值重建来分析结果。对曲线的分析给出了腔内质子渗透空间的有效介电常数和质子的扩散系数。通过Gilson等人（1985年，《分子生物学杂志》，183:503 - 516）给出的方程研究腔内的静电势。根据该分析，位点周围蛋白质的介电常数εprot≤6.5。质子在肌红蛋白 - 吡喃复合物血红素结合位点的扩散系数为D = 4×10(-5) cm2/s。该值约为质子在水中扩散系数的50%。较低的值表明腔内水的有序性增强，这一发现得到结合的大负熵ΔS0 = -46.6 cal·mol-1·deg-1的证实。通过动力学的数学重建研究了蛋白质中小腔保留质子的能力。已经证明，与能量耦合膜的Δψ一样大的库仑吸引力不足以将自由质子延迟到与质子产生位点的周转时间相当的时间范围内。
