Lu M, Balashov S P, Ebrey T G, Chen N, Chen Y, Menick D R, Crouch R K
Center for Biophysics and Computational Biology and Departments of Cell and Structural Biology, and Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Biochemistry. 2000 Mar 7;39(9):2325-31. doi: 10.1021/bi992554o.
Light absorbed by bacteriorhodopsin (bR) leads to a proton being released at the extracellular surface of the purple membrane. Structural studies as well as studies of mutants of bR indicate that several groups form a pathway for proton transfer from the Schiff base to the extracellular surface. These groups include D85, R82, E204, E194, and water molecules. Other residues may be important in tuning the initial state pK(a) values of these groups and in mediating light-induced changes of the pK(a) values. A potentially important residue is R134: it is located close to E194 and might interact electrostatically to affect the pK(a) of E194 and light-induced proton release. In this study we investigated effects of the substitution of R134 with a histidine on light-induced proton release and on the photocycle transitions associated with proton transfer. By measuring the light-induced absorption changes versus pH, we found that the R134H mutation results in an increase in the pK(a) of the proton release group in both the M (0.6 pK unit) and O (0.7 pK unit) intermediate states. This indicates the importance of R134 in tuning the pK(a) of the group that, at neutral and high pH, releases the proton upon M formation (fast proton release) and that, at low pH, releases the proton simultaneously with O decay (slow proton release). The higher pK(a) of the proton release group found in R134H correlates with the slowing of the rate of the O --> bR transition at low pH and probably is the cause of this slowing. The pH dependence of the fraction of the O intermediate is altered in R134H compared to the WT but is similar to that in the E194D mutant: a very small amount of O is present at neutral pH, but the fraction of O increases greatly upon decreasing the pH. These results provide further support for the hypothesis that the O --> bR transition is controlled by the rate of deprotonation of the proton release group. These data also provide further evidence for the importance of the R134-E194 interaction in modulating proton release from D85 after light has led to its being protonated.
细菌视紫红质（bR）吸收的光会导致质子在紫膜的细胞外表面释放。对bR的结构研究以及突变体研究表明，有几个基团形成了质子从席夫碱转移到细胞外表面的途径。这些基团包括D85、R82、E204、E194和水分子。其他残基可能在调节这些基团的初始状态pK(a)值以及介导光诱导的pK(a)值变化方面很重要。一个潜在重要的残基是R134：它位于E194附近，可能通过静电相互作用影响E194的pK(a)以及光诱导的质子释放。在本研究中，我们研究了用组氨酸取代R134对光诱导的质子释放以及与质子转移相关的光循环转变的影响。通过测量光诱导的吸收变化与pH的关系，我们发现R134H突变导致在M（0.6个pK单位）和O（0.7个pK单位）中间态下质子释放基团的pK(a)增加。这表明R134在调节该基团的pK(a)方面很重要，该基团在中性和高pH下，在M形成时释放质子（快速质子释放），而在低pH下，与O衰变同时释放质子（慢速质子释放）。在R134H中发现的质子释放基团的较高pK(a)与低pH下O→bR转变速率的减慢相关，可能是这种减慢的原因。与野生型相比，R134H中O中间体分数的pH依赖性发生了改变，但与E194D突变体中的相似：在中性pH下存在极少量的O，但随着pH降低，O的分数会大幅增加。这些结果为O→bR转变受质子释放基团去质子化速率控制这一假说提供了进一步支持。这些数据也进一步证明了R134 - E194相互作用在光使D85质子化后调节质子从D85释放中的重要性。
