Reply to Breuer et al.: Molecular dynamics simulations do not provide functionally relevant values of redox potential in MtrF
Reply to Breuer et al.: Molecular dynamics simulations do not provide functionally relevant values of redox potential in MtrF
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回复 Breuer 等人：分子动力学模拟未提供 MtrF 中氧化还原电位的功能相关值
DOI：
10.1073/pnas.1717048114
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Hiroshi Ishikita
中科院分区：
文献类型：
--
作者：
Hiroshi C. Watanabe;Yuki Yamashita;Hiroshi Ishikita
我们以前报道过Asp 631使血红素9的氧化还原电位（Em）降低136 mV [根据Breuer et al. (1)]在天然MtrF中，Asp 631质子化（对应于Asn 631突变）并不显著影响血红素9的Em，因为其他残基的去质子化补偿了Em位移（2）。Breuer等人(3)不能观察到相应的效果，因为他们固定了可滴定残基的质子化状态（1）。根据Breuer et al. (1)，残基对Em的贡献非常大;例如，Asp 228使血红素2的Em降低了− 2，280 mV [在我们的计算中为− 61 mV（2）]。在他们的信中，Breuer et al. (3)声明“比较单个残基贡献的符号而不是大小才有意义”;然而，在他们的原始报告中，他们不仅关注单个残基贡献的符号，而且关注单个残基贡献的大小，例如，声明“我们发现，在辅因子的环境中，每个带电残基都贡献了十分之几伏”（1）。每个残基对Em的贡献必须与突变后实验测量的Em位移相当。为了验证使用基于分子动力学（MD）模拟的热力学积分（TI）方法获得的Em，我们采用了广泛用于自由能计算的缓慢生长TI方法（4）（例如，参考文献5），其中氧化还原状态逐渐从Fe 3+转变为Fe 2+（2）。Breuer等人(3)反对我们的TI模拟的时间尺度（10 ns），但他们证明了他们的TI模拟的时间尺度（11 ns）假设结构“十血红素基序的刚度”（然而，见下文）。我们强调，任何基于MD的方法在这种情况下都是不合适的，无论模拟时间如何，原因如下。对于结构波动（图1），我们已经指出，在原始MtrF晶体结构的结构域I的β-桶基序中，侧链方向是不正确的（例如，疏水残基朝向本体溶剂），导致计算的B因子非常高（2）。然而，Breuer et al. (1)已经在MtrF晶体结构中使用了原始的侧链取向。血红素2最接近结构域I（血红素7最接近结构域III）。值得注意的是，只有血红素2和血红素7对的Em值在其完全对称的Em曲线中存在显著差异（130 mV）（1）。此外，甚至血红素结合结构域IV也是不稳定的（图1）。由于他们的模拟仍然处于域I和IV的缓慢衰减过程中，远离平衡，Em曲线强烈依赖于MD起始结构（例如，在平衡0，100和1，000 ns后获得），如我们的测试MD计算所示（2）。根据Breuer等人的“十血红素基序的相当大的刚性”的陈述，的字母（3），他们在计算Em（1）时忽略了这一点。最后，Breuer等人报道的血红素2（-57 mV）和7（74 mV）的Em。(1)似乎不太可能支持结合黄素[-150 mV（6）]作为末端电子受体的作用（7，8）。他们的MD simulationbased TI方法，使用它们的几何形状和固定的质子化状态的可滴定的残基和血红素-丙酸基团，是不可能提供功能相关的值的EM在MtrF。
We previously reported that Asp631 decreased the redox potential (Em) for heme 9 by 136 mV [1,362 mV according to Breuer et al.(1)] in native MtrF, whereas Asp631 protonation (corresponding to Asn631 mutation) did not significantly affect Em for heme 9, because deprotonation of other residues compensated for the Em shift (2). Breuer et al.(3) cannot observe the corresponding effect, since they fixed the protonation states of titratable residues (1). According to Breuer et al.(1), residues make unusually large contributions to Em; for example, Asp228 decreases Em for heme 2 by− 2,280 mV [− 61 mV in our calculations (2)]. In their letter, Breuer et al.(3) state that “it is only meaningful to compare the sign but not the magnitude of the single-residue contributions”; however, in their original report, they focus on not only the sign but also the magnitude of the single-residue contributions, stating, for example,“we find that every charged residue in the environment of a cofactor contributes several tenths of volts”(1). The contribution of each residue to Em must be comparable to Em shifts experimentally measured upon mutations. To verify Em obtained using their molecular dynamic (MD) simulation-based thermodynamic integration (TI) approach, we employed the slow-growth TI approach (4), as widely used for free energy calculations (eg, ref. 5), wherein the redox states gradually transit from Fe3+ to Fe2+(2). Breuer et al.(3) argue against the time scale of our TI simulations (10 ns), but they justify the time scale of their TI simulations (11 ns) assuming the structural “stiffness of the decaheme motif”(however, see below). We emphasize that any MD-based approaches are not appropriate in this case, irrespective of simulation time due to the following reasons. For the structural fluctuations (Fig. 1), we have pointed out that the side-chain orientations are incorrect in the β-barrel motif of domain I of the original MtrF crystal structure (eg, hydrophobic residues are oriented toward the bulk solvent), resulting in remarkably high calculated B-factors (2). Nevertheless, Breuer et al.(1) have used the original side-chain orientations in the MtrF crystal structure. Heme 2 is closest to domain I (heme 7 to domain III). Notably, only Em values of the heme 2 and heme 7 pair differ significantly (130 mV) in their perfectly symmetrical Em profile (1). Moreover, even heme-binding domain IV is also unstable (Fig. 1). Since their simulations were still in the slow-decay process of domains I and IV, being far from equilibrium, Em profiles strongly depend on the MD-starting structure (eg, obtained after equilibration for 0, 100, and 1,000 ns) as demonstrated in our test MD calculations (2). From the statement,“the considerable stiffness of the decaheme motif” in Breuer et al.’s letter (3), they have missed this point while calculating Em (1). Finally, the Em for hemes 2 (− 57 mV) and 7 (74 mV) reported by Breuer et al.(1) seem unlikely to support a role of bound flavin [− 150 mV (6)] serving as the terminal electron acceptor (7, 8). Their MD simulationbased TI approach, using their geometry and fixing the protonation states of titratable residues and heme-propionic groups, is unlikely to provide functionally relevant values of Em in MtrF.
水中空腔形成的热力学 - 分子动力学研究
DOI：
--
发表时间：
1982
期刊：
影响因子：
--
作者：
J. Postma;H. Berendsen;J. R. Haak
通讯作者：
J. R. Haak
影响因子：
6.6
作者：
Xu, Shuai;Jangir, Yamini;El-Naggar, Mohamed Y.
通讯作者：
El-Naggar, Mohamed Y.
影响因子：
56.9
作者：
BASH, PA;SINGH, UC;KOLLMAN, PA
通讯作者：
KOLLMAN, PA