Molecular Analysis of Terminal Oxidases from N2-fixing Azotobacter vinelandii

固氮固氮细菌末端氧化酶的分子分析

• 批准号: 9104280
• 负责人: Robert Maier
• 金额: $ 7.85万
• 依托单位: Johns Hopkins University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9104280&HistoricalAwards=false
• 关键词: Molecular; Analysis; Terminal; Oxidases; N2

Biological nitrogen fixation catalyzed by the procaryotic enzyme nitrogenase is essential for a positive input of N on Earth. The N2-fixing bacterium Azotobacter vinelandii contains two terminal oxidases responsible for aerobic O2-consuming respiration. One of the oxidases (cytochrome o) is coupled to ATP production, but the other oxidase (cytochrome b/d) complex) appears not to be coupled to energy generation. The latter vigorously O2-consuming oxidase is thought to be important in removing O2 so that the O2-labile nitrogenase enzyme can function. The specific roles of each of these oxidases in respiratory protection of nitrogenase and respiratory metabolism of the bacterium are not well-understood. It is proposed to compare structural gene mutants in the terminal oxidase cytochrome b/d and cytochrome o with the wild type in many parameters associated with N2 fixation respiration. Also the cyt b/d complex will be purified and reconstituted into lipid vesicles to directly measure its ability to generate an electrochemical proton gradient (energy-generating ability). Assuming lack of electrochemical gradient formation by the purified cyt b/d complex in membranes, we will then characterize the pertinent structural features that span the periplasmic and cytoplasmic portions of the membrane, and responsible for differences in the uncoupled (non energy generating) terminal oxidase of A. vinelandii in comparison with the well-characterized energy-coupled cyt b/d oxidase of E. coli. The transcriptional regulation of these two terminal oxidase complexes, especially their regulation in response to N and O2 levels will be addressed. The results are expected to enhance our knowledge of the diversity and flexibility of bacterial respiratory metabolism and specifically on respiratory metabolism in conjunction with N2 fixation.

由原核生物固氮酶催化的生物固氮是地球上氮的正输入所必需的。固定氮气的棕色固氮细菌含有两种末端氧化酶，负责好氧消耗氧气的呼吸。其中一种氧化酶(细胞色素o)与三磷酸腺苷的产生有关，但另一种氧化酶(细胞色素b/d复合体)似乎与能量的产生无关。后者强烈地消耗氧气，被认为在清除氧气方面很重要，这样不稳定氧气的固氮酶才能发挥作用。这些氧化酶在固氮酶的呼吸保护和细菌的呼吸代谢中的具体作用还不是很清楚。建议将末端氧化酶、细胞色素b/d和细胞色素o的结构基因突变体与野生型在许多与氮固定呼吸相关的参数上进行比较。此外，细胞色素b/d复合体将被提纯并重组为脂泡，以直接测量其产生电化学质子梯度的能力(产生能量的能力)。假设纯化的细胞色素b/d复合体在膜中缺乏电化学梯度的形成，那么我们将表征跨越膜的周质和细胞质部分的相关结构特征，并与特征良好的能量耦合的细胞色素b/d氧化酶相比，导致棕色拟青霉的非偶联(非产能)末端氧化酶的差异。这两个末端氧化酶复合体的转录调节，特别是它们对N和O2水平的调节将被讨论。这些结果有望增强我们对细菌呼吸代谢的多样性和灵活性的了解，特别是结合氮气固定的呼吸代谢。