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

在富含氨的环境中(如肥田和污水处理厂)，某些类型的细菌使用含铁的血红素酶将固定氮转化为一氧化二氮(N2O)--一种强有力的消耗臭氧的温室气体。该项目将研究基于血红素的N2O形成的分子基础；这一知识与开发更有效的农业实践以及减少大气污染有关。通过研究分子结构对血红素催化N2O生成的影响，该项目还将为生物技术合成酶的设计提供参考。本科生研究是这个项目的一个关键特点，为学生提供技能、经验和未来科学职业道路的动力。细胞色素p460与羟胺反应生成N2O的方法最近引起了人们的关注，重点是来自硝化细菌的酶。相比之下，人们对甲烷氧化菌中的等价酶知之甚少。本项目将集中于两种进化上相关的C型血红素蛋白的结构-功能关系：1)羟胺氧化细胞色素p460(McP460)；2)一氧化氮(NO)结合细胞色素c‘-β(McCP-beta)。这些蛋白质的晶体结构显示了共同的三级结构，但远端的血红素口袋非常不同；McP460包含一个赖氨酸-卟啉交联剂和一个为酶功能量身定做的高极性的远端口袋，而McCP-beta已经进化出一个疏水的远端血红素环境，不需要结合。具体目标是：(I)表征McP460催化的羟胺氧化和N2O形成的反应中间产物，以及p460赖氨酸-卟啉交联键和远端口袋氢键残基的影响；(Ii)确定McCP-β的作用，以及与其从N2O生成到其拟议的非结合作用的功能演变相关的血红素化学。一系列物理化学技术将被用于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、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-质谱学、X射线结晶学、密度泛函理论和电位测量。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.