Madeline E Rodemeier, Olivia P Holsinger, Andrew R Buller
{"title":"钴取代血红蛋白表达。","authors":"Madeline E Rodemeier, Olivia P Holsinger, Andrew R Buller","doi":"10.1016/bs.mie.2025.06.031","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"720 ","pages":"55-76"},"PeriodicalIF":0.0000,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Cobalt-substituted hemoprotein expression.\",\"authors\":\"Madeline E Rodemeier, Olivia P Holsinger, Andrew R Buller\",\"doi\":\"10.1016/bs.mie.2025.06.031\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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.</p>\",\"PeriodicalId\":18662,\"journal\":{\"name\":\"Methods in enzymology\",\"volume\":\"720 \",\"pages\":\"55-76\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2025-01-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Methods in enzymology\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1016/bs.mie.2025.06.031\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2025/7/4 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q3\",\"JCRName\":\"Biochemistry, Genetics and Molecular Biology\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Methods in enzymology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1016/bs.mie.2025.06.031","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/7/4 0:00:00","PubModel":"Epub","JCR":"Q3","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
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.
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.