Esquerra Raymond M, Jensen Russell A, Bhaskaran Shyam, Pillsbury Marlisa L, Mendoza Juan L, Lintner Benjamin W, Kliger David S, Goldbeck Robert A
Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California 94132, USA.
J Biol Chem. 2008 May 16;283(20):14165-75. doi: 10.1074/jbc.M709710200. Epub 2008 Mar 20.
We monitored the occupancy of a functionally important non-coordinated water molecule in the distal heme pocket of sperm whale myoglobin over the pH range 4.3-9.4. Water occupancy was assessed by using time-resolved spectroscopy to detect the perturbation of the heme visible band absorption spectrum caused by water entry after CO photodissociation ( Goldbeck, R. A., Bhaskaran, S., Ortega, C., Mendoza, J. L., Olson, J. S., Soman, J., Kliger, D. S., and Esquerra, R. M. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 1254-1259 ). We found that the water occupancy observed during the time interval between ligand photolysis and diffusive recombination decreased by nearly 20% as the pH was lowered below 6. This decrease accounted for most of the concomitant increase in the observed CO bimolecular recombination rate constant, as the lower water occupancy presented a smaller kinetic barrier to CO entry into the pocket at lower pH. These results were consistent with a model in which the distal histidine, which stabilizes the water molecule within the distal pocket by accepting a hydrogen bond, tends to swing out of the pocket upon protonation and destabilize the water occupancy at low pH. Extrapolation of this model to lower pH suggests that the additional increase in ligand association rate constant observed previously in stopped-flow studies at pH 3 may also be due in part to reduced distal water occupancy concomitant with further His64 protonation and coupled protein conformational change.
我们监测了抹香鲸肌红蛋白远侧血红素口袋中一个功能重要的非配位水分子在pH值4.3 - 9.4范围内的占有率。通过时间分辨光谱法检测CO光解离后水分子进入引起的血红素可见带吸收光谱的扰动来评估水的占有率（Goldbeck, R. A., Bhaskaran, S., Ortega, C., Mendoza, J. L., Olson, J. S., Soman, J., Kliger, D. S., and Esquerra, R. M. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 1254 - 1259）。我们发现，当pH值降至6以下时，在配体光解和扩散重组之间的时间间隔内观察到的水占有率下降了近20%。这种下降占观察到的CO双分子重组速率常数同时增加的大部分原因，因为较低的水占有率在较低pH值下对CO进入口袋呈现出较小的动力学障碍。这些结果与一个模型一致，在该模型中，通过接受氢键来稳定远侧口袋内水分子的远侧组氨酸，在质子化时倾向于摆动出该口袋，并在低pH值下使水占有率不稳定。将该模型外推到更低的pH值表明，先前在pH值为3的停流研究中观察到的配体缔合速率常数的额外增加，也可能部分归因于随着His64进一步质子化和相关蛋白质构象变化而导致的远侧水占有率降低。
