Institute of Biophysical Chemistry & Centre for Biomolecular Magnetic Resonance, Goethe-University Frankfurt, Frankfurt, Germany.
J Am Chem Soc. 2011 Mar 30;133(12):4645-54. doi: 10.1021/ja111116a. Epub 2011 Mar 2.
The proteorhodopsin (PR) family found in bacteria near the ocean's surface consists of hundreds of PR variants color-tuned to their environment. PR contains a highly conserved single histidine at position 75, which is not found in most other retinal proteins. Using (13)C and (15)N MAS NMR, we were able to prove for green PR that His75 forms a pH-dependent H-bond with the primary proton acceptor Asp97, which explains its unusually high pK(a). The functional role of His75 has been studied using site-directed mutagenesis and time-resolved optical spectroscopy: Ultrafast vis-pump/vis-probe experiments on PR(H75N) showed that the primary reaction dynamics is retained, while flash photolysis experiments revealed an accelerated photocycle. Our data show the formation of a pH-dependent His-Asp cluster which might be typical for eubacterial retinal proteins. Despite its stabilizing function, His75 was found to slow the photocycle in wild-type PR. This means that PR was not optimized by evolution for fast proton transfer, which raises questions about its true function in vivo.
在海洋表面附近的细菌中发现的细菌视紫红质（PR）家族由数百种对其环境进行颜色调谐的 PR 变体组成。PR 含有一个高度保守的单个组氨酸残基，位于第 75 位，而在大多数其他视蛋白中不存在。使用（13）C 和（15）N MAS NMR，我们能够证明绿色 PR 中的 His75 与主要质子受体 Asp97 形成 pH 依赖性氢键，这解释了其异常高的 pK（a）。通过定点突变和时间分辨光光谱学研究了 His75 的功能作用：对 PR（H75N）进行超快可见泵浦/可见探针实验表明，保持了主要的反应动力学，而闪光光解实验则揭示了加速的光循环。我们的数据表明形成了一个 pH 依赖性的 His-Asp 簇，这可能是真细菌视蛋白的典型特征。尽管 His75 具有稳定作用，但在野生型 PR 中发现它会减慢光循环。这意味着 PR 不是通过进化来优化快速质子转移的，这引发了关于其在体内真正功能的问题。
