Cobalt-substituted hemoprotein expression.

4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-07-04 DOI:10.1016/bs.mie.2025.06.031
Madeline E Rodemeier, Olivia P Holsinger, Andrew R Buller
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引用次数: 0

Abstract

Substituting the native metal of metalloenzymes can significantly alter the enzymes' reactivity and spectroscopic properties. Cobalt is especially attractive as a substitute for the native iron center in hemoproteins, as it generates metal variants with complementary spectroscopic properties and could enable new modes of reactivity. Here, we describe a detailed protocol for the biosynthesis and incorporation of cobalt protoporphyrin IX (CoPPIX) into hemoproteins, replacing the native heme b cofactor during expression in the common laboratory strain Escherichia coli BL21(DE3). This protocol is described using the model hemoprotein Physeter macrocephalus (sperm whale) myoglobin. Because of cobalt's unique electronic and geometric properties, cobalt-substituted hemoproteins offer a valuable handle for spectroscopic characterization and structural studies. We describe analytic methods of assessing cofactor identity and purity, including electronic absorption spectroscopy, liquid-chromatography/mass-spectrometry, inductively coupled plasma-mass spectrometry, and electron paramagnetic resonance spectroscopy. This method for generating artificial metalloenzymes is effective, easy to implement, and can produce useful quantities of Co-substituted hemoproteins.

钴取代血红蛋白表达。
取代金属酶的天然金属可以显著改变酶的反应性和光谱性质。钴作为血红蛋白中天然铁中心的替代品尤其具有吸引力,因为它可以产生具有互补光谱特性的金属变体,并可以实现新的反应模式。在这里,我们描述了一个详细的方案,钴原卟啉IX (CoPPIX)的生物合成和整合到血红蛋白,取代天然血红素b辅助因子在普通实验室菌株大肠杆菌BL21(DE3)的表达过程中。该方案是用模型血蛋白Physeter macrocephalus(抹香鲸)肌红蛋白描述的。由于钴独特的电子和几何特性,钴取代血红蛋白为光谱表征和结构研究提供了有价值的处理方法。我们描述了评估辅助因子身份和纯度的分析方法,包括电子吸收光谱、液相色谱/质谱、电感耦合等离子体质谱和电子顺磁共振光谱。这种合成人工金属酶的方法是有效的,容易实现的,并且可以产生有用数量的共取代血红蛋白。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Methods in enzymology
Methods in enzymology 生物-生化研究方法
CiteScore
2.90
自引率
0.00%
发文量
308
审稿时长
3-6 weeks
期刊介绍: The critically acclaimed laboratory standard for almost 50 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Each volume is eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with over 500 volumes the series contains much material still relevant today and is truly an essential publication for researchers in all fields of life sciences, including microbiology, biochemistry, cancer research and genetics-just to name a few. Five of the 2013 Nobel Laureates have edited or contributed to volumes of MIE.
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