RUI: Functional Evolution of Cytochrome P460 and Cytochrome c'-beta

RUI：细胞色素 P460 和细胞色素 c-beta 的功能进化

• 批准号: 1921670
• 负责人: Colin Andrew
• 金额: $ 35.69万
• 依托单位: Eastern Oregon University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1921670&HistoricalAwards=false
• 关键词: RUI; Functional; Evolution; Cytochrome; P460

In ammonia-rich environments (e.g. fertilized fields and wastewater treatment plants), certain types of bacteria use iron-containing heme enzymes to convert fixed nitrogen to nitrous oxide (N2O)- a potent ozone-depleting greenhouse gas. This project will study the molecular basis for heme-based N2O formation; this knowledge is relevant for developing more efficient agricultural practices along with reducing atmospheric pollution. By studying the influence of molecular structure on heme-catalyzed N2O formation, this project will also inform the design of synthetic enzymes for biotechnology. Undergraduate research is a key feature of this project, providing students with skills, experiences and motivation for future scientific career pathways. The means by which cytochromes P460 react with hydroxylamine to produce N2O has received recent attention, with a focus on enzymes from nitrifying bacteria. By contrast, little is known about the equivalent enzymes in methanotrophs. This project will focus on structure-function relationships in two evolutionarily related c-type heme proteins from the methanotroph, Methylococcus capsulatus (Bath): 1) a hydroxylamine oxidizing cytochrome P460 (McP460); and 2) a nitric oxide (NO)-binding cytochrome c'-beta (McCP-beta). Crystal structures of these proteins reveal common tertiary structures, but very different distal heme pockets; McP460 contains a Lys-porphyrin cross-link and a highly polar distal pocket tailored to enzymatic function, whereas McCP-beta has evolved a hydrophobic distal heme environment for NO binding. Specific aims are to (i) characterize reaction intermediates in McP460-catalyzed hydroxylamine oxidation and N2O formation, along with the influence of the P460 Lys-porphyrin cross-link and distal pocket hydrogen-bonding residues; and (ii) define the role of the McCP-beta 'distal Phe cap', as well as heme chemistry pertinent to its functional evolution from N2O generation to its proposed NO-binding role. A battery of physicochemical techniques will be utilized to study wild-type and variant proteins, including spectroscopy (UV-visible absorption, resonance Raman, FT infra-red, electron paramagnetic resonance), kinetic techniques (stopped-flow, freeze-quench and flash photolysis), as well as gas chromatography-mass spectrometry, X-ray crystallography, density functional theory, and potentiometric measurements.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

在富含氨的环境中（例如施肥的田地和废水处理厂），某些类型的细菌使用含铁血红素酶将固定氮转化为一氧化二氮（N2 O）-一种强效的臭氧消耗温室气体。该项目将研究血红素N2 O形成的分子基础;这一知识与发展更有效的农业实践沿着减少大气污染有关。通过研究分子结构对血红素催化N2 O形成的影响，该项目还将为生物技术合成酶的设计提供信息。本科生研究是该项目的一个关键特征，为学生提供未来科学职业道路的技能，经验和动力。细胞色素P460与羟胺反应产生N2 O的方法最近受到关注，重点是硝化细菌的酶。相比之下，对甲烷氧化菌中的等效酶知之甚少。该项目将重点关注甲烷氧化菌Methylococcus capsulatus（Bath）中两种进化相关的c型血红素蛋白的结构-功能关系：1）羟胺氧化细胞色素P460（McP 460）;和2）一氧化氮（NO）结合细胞色素c '-β（McCP-β）。这些蛋白质的晶体结构揭示了共同的三级结构，但非常不同的远端血红素口袋; McP 460含有赖氨酸-卟啉交联和针对酶功能定制的高极性远端口袋，而McCP-β已经进化出用于NO结合的疏水远端血红素环境。具体目标是（i）表征McP 460催化的羟胺氧化和N2 O形成中的反应中间体，沿着P460赖氨酸-卟啉交联和远端口袋氢键残基的影响;和（ii）定义McCP-β“远端Phe帽”的作用，以及与其从N2 O生成到其拟议的NO结合作用的功能演变相关的血红素化学。一系列的物理化学技术将被用来研究野生型和变异蛋白质，包括光谱学（紫外-可见吸收、共振拉曼、FT红外、电子顺磁共振）、动力学技术（停流、冷冻猝灭和闪光光解），以及气相色谱-质谱法、X射线晶体学、密度泛函理论，该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Hydroxylamine Complexes of Cytochrome c′ : Influence of Heme Iron Redox State on Kinetic and Spectroscopic Properties

细胞色素 c 的羟胺配合物：血红素铁氧化还原状态对动力学和光谱性质的影响

• DOI: 10.1021/acs.inorgchem.0c01925
• 发表时间: 2020
• 期刊: Inorganic Chemistry
• 影响因子: 4.6
• 作者: Brown, Brianna N.;Robinson, Kelsey J.;Durfee, Quentin C.;Kekilli, Demet;Hough, Michael A.;Andrew, Colin R.
• 通讯作者: Andrew, Colin R.