Friedman N, Gat Y, Sheves M, Ottolenghi M
Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel.
Biochemistry. 1994 Dec 13;33(49):14758-67. doi: 10.1021/bi00253a014.
The M stage in the photocycle of bacteriorhodopsin (bR), a key step in its light-induced proton pump mechanism, is studied in water/glycerol suspensions over the temperature range between 20 and -60 degrees C. The biexponential decay of M is analyzed for wild-type (WT) bR and for its D96N, Y185F, and D115N mutants, at various pH values, according to the scheme: bR-->(hv) L-->M<-->(k1, k-1) N-->(k2) bR. The analysis leads to the conclusion that the N state is generated, with analogous rate parameters, in all cases, including the D96N mutant. Another approach involves probing the M state, generated by steady-state illumination at -60 degrees C, by fast cooling to -180 degrees C. Subsequent irradiation with blue light, followed by gradual warming up, induces the M-->(hv) M'-->bR'-->bR sequence of reactions. On the basis of characteristic difference spectra and transition temperatures observed for the M'-->bR' process, it is concluded that the initially observed M state at -60 degrees C, denoted as (M)a, is composed of three (or four) equilibrated substrates, MI, MII, MIII, and MIV. During the M-->N equilibration, which corresponds to the fast phase of the M decay, (M)a transforms into a second state, (M)b, in which MIII has been replaced by a fifth M substate, denoted as MV. MV is identified as the protein state in which an appropriate structural change allows reprotonation of the Schiff base, generating the N state. The low-temperature heterogeneity in M is discussed in terms of the two M states (M1 and M2) previously postulated [Váró, G., & Lanyi, J. K. (1990) Biochemistry 29, 2241] for the room temperature photocycle. The following conclusions are derived for both low and room temperature photocycles: (a) The M population is highly heterogeneous and pH dependent. (b) At least three transitions are observed between the initially formed M state and the M state that is equilibrated with N. These are assigned to protein conformational changes and to water molecule rearrangements. (c) In an aqueous suspension of WT bR at room temperature, the Schiff base reprotonation is controlled by D96. However, our results show that the formation and stability of the N state do not require the D96 residue. Moreover, at low temperatures, the (M)a-->(M)b protein structural transformation, which has not yet been resolved at room temperature, becomes the rate-determining step in the protonation of the Schiff base.
细菌视紫红质（bR）光循环中的M阶段是其光诱导质子泵机制的关键步骤，本文在20至 -60摄氏度的温度范围内，对水/甘油悬浮液中的该阶段进行了研究。根据以下反应式：bR-->(hv) L-->M<-->(k1, k-1) N-->(k2) bR，分析了野生型（WT）bR及其D96N、Y185F和D115N突变体在不同pH值下M的双指数衰减。分析得出的结论是，在所有情况下，包括D96N突变体，都会以类似的速率参数生成N状态。另一种方法是通过快速冷却至 -180摄氏度来探测在 -60摄氏度下通过稳态光照产生的M状态。随后用蓝光照射，然后逐渐升温，会诱导出M-->(hv) M'-->bR'-->bR的反应序列。根据观察到的M'-->bR'过程的特征差异光谱和转变温度，得出结论：在 -60摄氏度下最初观察到的M状态，记为(M)a，由三个（或四个）平衡的底物MI、MII、MIII和MIV组成。在与N平衡的M-->N过程中，这对应于M衰减的快速阶段，(M)a转变为第二种状态(M)b，其中MIII已被第五个M亚状态取代，记为MV。MV被确定为蛋白质状态，其中适当的结构变化允许席夫碱重新质子化，从而产生N状态。本文根据先前为室温光循环假设的两种M状态（M1和M2）讨论了M中的低温异质性。对于低温和室温光循环都得出了以下结论：(a) M群体高度异质且依赖于pH值。(b) 在最初形成的M状态和与N平衡的M状态之间观察到至少三个转变。这些转变归因于蛋白质构象变化和水分子重排。(c) 在室温下WT bR的水悬浮液中，席夫碱的重新质子化由D96控制。然而，我们的结果表明，N状态的形成和稳定性不需要D96残基。此外，在低温下，(M)a-->(M)b蛋白质结构转变（在室温下尚未解析）成为席夫碱质子化的速率决定步骤。
