Molecular and Biochemical Basis for Stable Carbon Isotope Ratios in Marine Autotrophs using Form IA RubisCO

稳定碳同位素数据在生态学和地球科学中有着广泛的应用。这些关于同位素分馏的数据影响了我们对从分子到全球尺度上发生的过程的理解。过去气候的后遗症，限制当今温室气体排放命运的模型，复杂生态系统内营养转移的推断，以及酶和细胞对初级生产力控制的阐明，都使用了稳定的碳同位素分馏数据(增量13C值)。尽管这种分馏信息具有广泛的适用性，但这些数据几乎总是被解释为假设碳是固定的，其同位素被分馏，就好像菠菜的鲁比斯科(ribulose?1，5?bisphosphate?carboxylase/oxygenase)负责！这可能是一个巨大的错误，并可能实质上使所有后续对上述问题的数据解释无效。在已研究的少数情况下，RuBisCO的动力学同位素效应(Kie)，即固定碳的13C与所用的二氧化碳底物的增量13C不同的程度，已被证明差异很大。因此，确定不同RuBisCO的Kie对于理解、解释三角洲13C以及在海洋学中建立三角洲13C值的模型至关重要。因此，确定自养生物中所有形式的Rubisco的Kie是一个长期的研究目标。为了确定IA形式RuBisCOs的Kies，我们将从具有代表性的海洋微微型浮游植物和化学自养细菌中分离和纯化这些固碳酶。将使用模式种聚球藻WH7803、原氯球菌MED 4和两种硫氧化化学自养细菌、海岸和喷口双壳类的共生体Solemya velum和Bathyhemolus thermophilus。分离的蛋白质将根据氨基酸序列、组成、Vmax和Km(CO2)进行表征。最后，将用高精度的方法测定动力学同位素效应。确定IA形式RuBisCOs的KIE将使我们能够评估三角洲13C分析的基本假设，并在精度大幅提高的情况下推断影响海洋初级生产力的因素。

Stable carbon isotope data are used widely in ecology and earth sciences. These data on isotope fractionations influence our understandings of processes occurring on molecular to global scales. Hindcasts of past climates, models constraining the fates of present day greenhouse gas emissions, inferences of trophic transfers within complex ecosystems, and elucidation of enzyme and cellular controls on primary productivity have all used stable carbon isotope fractionation data (delta 13C values). Despite the wide applicability of such fractionation information, such data are nearly always interpreted assuming carbon is fixed and its isotopes fractionated as if the ribulose?1,5?bisphosphate?carboxylase/oxygenase (RubisCO) of spinach was responsible! This is probably a huge mistake and may substantially invalidate all of the subsequent data interpretations for problems such as those mentioned above.In the few cases that have been studied, the kinetic isotope effects (KIEs) forRubisCOs, the degree to which the delta 13C of the carbon fixed differs from the C02 substrate used, have been demonstrated to vary broadly. Thus, determining the KIEs of different RubisCOs is critical to understanding, delta 13C interpretation, and modeling of delta 13C values in oceanography. As a result, it is a long?term research goal to determine KIEs from all forms of RubisCO in autotrophs. In order to determine the KIEs of Form IA RubisCOs, these carbon-fixing enzymes will be isolated and purified from representative marine picophytoplankton and chemoautotrophic bacteria. Model species Synechococcus WH7803, Prochlorococcus MED 4, and two sulfur?oxidizing chemoautotrophs, symbionts of the coastal and vent bivalves, Solemya velum and Bathymodiolus thermophilus will be used. The isolated proteins will be characterized with respect to amino acid sequence, composition, Vmax, and Km (CO2). Finally, the kinetic isotope effects will be determined by high precision methods. Determining the KIEs for Form IA RubisCOs will allow us to evaluate a fundamental assumption of delta 13C analyses, and to infer the factors affecting oceanic primary productivity with a substantial improvement in accuracy.