Pathway of proton transfer in bacterial reaction centers: second-site mutation Asn-M44-->Asp restores electron and proton transfer in reaction centers from the photosynthetically deficient Asp-L213-->Asn mutant of Rhodobacter sphaeroides.
Pathway of proton transfer in bacterial reaction centers: second-site mutation Asn-M44-->Asp restores electron and proton transfer in reaction centers from the photosynthetically deficient Asp-L213-->Asn mutant of Rhodobacter sphaeroides.
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细菌反应中心质子转移途径：第二位点突变 Asn-M44-->Asp 恢复了球红杆菌光合缺陷的 Asp-L213-->Asn 突变体在反应中心的电子和质子转移。
DOI：
10.1073/pnas.90.4.1325
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发表时间：
1993
影响因子：
11.1
通讯作者：
Okamura,MY
中科院分区：
文献类型：
--
作者：
Rongey,SH;Paddock,ML;Feher,G;Okamura,MY
对球形红杆菌（Rhodobacter sphaeroides）光合反应中心（RC）的位点定向诱变研究表明，L亚基（Asp-L213）的Asp-213对光合活性起重要作用。Asn取代Asp-L213导致了一个光合缺陷突变体，这是由于质子耦合电子转移反应QA-QB- + 2H+—>QAQBH2 （k(2)AB）的速度慢了10(4)倍。Asn- l213的有害作用是令人惊讶的，因为来自绿红假单胞菌、红红螺旋菌和金绿藻的RCs在同源位置上有Asn。然而，来自这些细菌的RCs具有一个Asp位于QB（二级醌受体）附近，与Rb中的Asn-M44同源。可以代替Asp-L213的球形金属。为了验证这一猜想，在Rb中引入了一个“病毒样”结构。用Asn代替Asp- l213，用Asp代替Asn- m44。该双突变体的RCs在电子转移反应k(2)AB中表现出接近原生的速率，并恢复了光合能力。第一电子转移反应QA-QB—>QAQB- (k(1)AB)和电荷重组D+QAQB—>DQAQB （kBD）的速率也恢复到接近原生值。这些结果表明，在QB附近的L213或M44位点上的Asp都可以提供快速质子转移的途径，并解释了为什么Asp-L213在不同的光合细菌中不需要保守。为了进一步测试M44位点的Asp对QB电子和质子转移的影响，构建了一个同时含有M44位点和L213位点Asp的突变体。该突变体（Asn-M44—>Asp）的RCs表现出更快的质子耦合电子向QB-的转移。在QB附近存在带负电荷的Asp残基时，质子耦合电子转移速率（k(2)AB）的增加表明，QB附近的Asp作为(i)质子转移链中的质子供体基团和/或（ii）带负电荷的残基稳定质子向还原QB的转移。
Site-directed mutagenesis of the photosynthetic reaction center (RC) from Rhodobacter sphaeroides has shown Asp-213 of the L subunit (Asp-L213) to be important for photosynthetic viability. Replacement of Asp-L213 with Asn resulted in a photosynthetically deficient mutant, due to the 10(4)-fold slower rate for the proton-coupled electron transfer reaction QA-QB- + 2H+-->QAQBH2 (k(2)AB). The detrimental effect of Asn-L213 is surprising since RCs from Rhodopseudomonas viridis, Rhodospirillum rubrum, and Chloroflexus aurantiacus have Asn at the homologous position. However, RCs from these bacteria have an Asp located near QB (the secondary quinone acceptor) at the position homologous to Asn-M44 in Rb. sphaeroides which might function in place of Asp-L213. To test this conjecture a "viridis-like" structure was introduced into Rb. sphaeroides by replacing Asp-L213 with Asn and Asn-M44 with Asp. The RCs from this double mutant displayed near-native rates for the electron transfer reaction k(2)AB and restored photosynthetic competence. The rates for the first electron transfer reaction QA-QB-->QAQB- (k(1)AB) and charge recombination D+QAQB--->DQAQB (kBD) were also restored to near-native values. These results indicate that Asp at either the L213 or the M44 site near QB can provide a pathway for rapid proton transfer and explain why Asp-L213 need not be conserved in different photosynthetic bacteria. To test further the effect of Asp at M44 on electron and proton transfer to QB a mutant containing Asp at both L213 and M44 was constructed. The RCs from this mutant (Asn-M44-->Asp) exhibited faster proton-coupled electron transfer to QB-. The increased rate of proton-coupled electron transfer (k(2)AB) in the presence of negatively charged Asp residues near QB suggests the role of an Asp near QB as (i) a proton donor group in the proton transfer chain and/or (ii) a negatively charged residue stabilizing proton transfer to reduced QB.