Role of Atypical D1 Proteins in Photosystem II

在光合作用中，光被用来从水中除去氢以产生氧。然后使用氢当量将二氧化碳化学还原为有机分子。水的氧化反应是由一种称为光系统II（PSII）的酶催化的。光很难催化这种反应，到目前为止，唯一已知的有效系统是自然系统。反应在含有4个锰离子和1个钙离子的离子交换剂中进行。该簇被PSII中被称为“D1蛋白”的高度保守的蛋白质结合并稳定。PSII复合物包含20多个其他蛋白质亚基。然而，在活跃的叶子中，D1蛋白质降解得非常快，大约每30分钟降解一次。这被认为是因为水裂解反应产生的活性氧副产物破坏了蛋白质。一个复杂的修复系统存在，以再生PSII与新鲜的D1蛋白。蓝细菌有几种不同的D1基因，适合不同的情况。有些是在强光下合成的，有些是在低氧环境中合成的。一些光合作用的蓝细菌也可以固定空气中的氮。这可能是一个问题，因为固氮酶被氧气不可逆地抑制。这些生物要么将固氮酶从光合作用中物理分离出来，要么只在夜间PSII不活跃时固氮。最近发现的一类D1基因相对于“典型D1”在结合锰簇的位点发生突变。这些突变使得这些“流氓”D1被认为不能析氧，然而，有足够的官能团用于金属结合是可能的。我们认为，非典型的D1序列可能是一种机制，以使PSII与非催化的D1，然后更换时，需要再次活动。还有一类甚至更不典型的D1（“超级流氓D1”），与蓝细菌有关，蓝细菌通过制造红移叶绿素，叶绿素f来适应远红光。这种叶绿素在红外线中具有峰值吸收，但似乎仍然能够使用这些较低能量的光子来驱动水氧化。超流氓D1在远红光条件下上调1000倍，因此可能在适应远红光中起作用。非典型D1序列在遗传学上是早期的，因此让人想起祖先D1，因此可以提供关于产氧光合作用进化的信息。如果在底物氧化中起作用，则变体PSII可以使用除水以外的底物。有趣的是它本身也为光合作用的进化提供了新的视角。我们将在体内和体外研究非典型D1蛋白在PSII中的功能。在体内研究中，我们将在各种条件下培养具有非典型D1的蓝藻，包括昼夜光暗节律。我们将研究与正常D1相比，非典型D1的表达模式与其他因素（如光暗、固氮和外部碳源）的关系。对于体外研究，我们将用流氓和超级流氓D1纯化PSII。我们将研究它们的功能，如金属含量，氧化底物和转移电子的能力。我们将评估所有的PSII亚基在修改后的反应中心的存在。通过结合体内和体外方法，我们将了解非典型D1序列的生物学功能是什么，以及如何在生物化学和生物物理水平上完成。

In photosynthesis, light is used to remove the hydrogen from water to give oxygen. The hydrogen equivalents are then used to chemically reduce carbon dioxide to organic molecules. The water oxidation reaction is catalysed by an enzyme known as photosystem II (PSII). It is hard to catalyse this reaction with light, so far the only known effective system is the natural one. The reaction takes place at a metallocluster containing 4 manganese ions and a calcium ion. This cluster is bound and stabilised by a highly conserved protein known as the "D1 protein" in PSII. The PSII complex contains more than 20 other protein subunits. However, in an active leaf, the D1 protein is degraded extremely quickly, every 30 minutes or so. This is thought to be because of the generation of reactive oxygen side products from the water-splitting reaction which damage the protein. A sophisticated system of repair exists to regenerate PSII with fresh D1 protein. Cyanobacteria have several different D1 genes suited to different situations. Some are synthesized in response to high light, others in low oxygen environments. Some photosynthetic cyanobacteria can also fix nitrogen from the air. This can be a problem as the nitrogenase enzyme is irreversibly inhibited by oxygen. These organisms either physically separate the nitrogenase from photosynthesis, or only fix nitrogen at night when PSII is inactive.There is a recently discovered a class of D1 genes that are mutated relative to the "canonical D1" at the sites binding the manganese cluster. The mutations are such that these "rogue" D1 are not thought to be capable of oxygen evolution, however, there are sufficient functional groups for metal binding to be a possibility. We believe that the atypical D1 sequences might be a mechanism to inactivate PSII with a non-catalytic D1, which is then replaced when activity is required again. There is a further class of even more atypical D1 ("super-rogue D1") that is associated with cyanobacteria that adapt to far-red light by making a red-shifted chlorophyll, chlorophyll f. This chlorophyll has a peak absorption in the infra-red, yet seems to still be capable of using these lower energy photons to drive water oxidation. The super-rogue D1 is 1000-fold up-regulated in far-red light conditions, so probably has a role in adaptation to far-red light.The atypical D1 sequences are phylogenetically early, so are reminiscent of an ancestral D1, so could provide information on the evolution of oxygenic photosynthesis. If functional in substrate oxidation, the variant PSII may use a substrate other than water. Of interest in itself and again providing insight into the evolution of photosynthesis. Such a reaction centre would be a novel finding.We will investigate the function of the atypical D1 proteins in PSII both in vivo and in vitro. For in vivo studies we will culture cyanobacteria with atypical D1 under a variety of conditions, including circadian light-dark rhythms. We will investigate the expression pattern of the atypical D1 in comparison to normal D1, in relation to other factors, such as light-dark, nitrogen fixation, and external carbon sources. For in vitro studies we will purify PSII with the rogue and super-rogue D1. We will investigate their function, such as metal content, ability to oxidise substrates and transfer electrons. We will assess the presence of all of the PSII subunits in the modified reaction centres. With a combination of the in vivo and in vitro approaches we will learn what the biological function of the atypical D1 sequences is, and how, at a biochemical and biophysical level, it is accomplished.