Enzymes that bear a nonnative or artificially introduced metal center can engender novel reactivity and enable new spectroscopic and structural studies. In the case of metal-organic cofactors, such as metalloporphyrins, no general methods exist to build and incorporate new-to-nature cofactor analogs in vivo. We report here that a common laboratory strain, Escherichia coli BL21(DE3), biosynthesizes cobalt protoporphyrin IX (CoPPIX) under iron-limited, cobalt-rich growth conditions. In supplemented minimal media containing CoCl₂, the metabolically produced CoPPIX is directly incorporated into multiple hemoproteins in place of native heme b (FePPIX). Five cobalt-substituted proteins were successfully expressed with this new-to-nature cobalt porphyrin cofactor: myoglobin H64V V68A, dye decolorizing peroxidase, aldoxime dehydratase, cytochrome P450 119, and catalase. We show conclusively that these proteins incorporate CoPPIX, with the CoPPIX making up at least 95% of the total porphyrin content. In cases in which the native metal ligand is a sulfur or nitrogen, spectroscopic parameters are consistent with retention of native metal ligands. This method is an improvement on previous approaches with respect to both yield and ease-of-implementation. Significantly, this method overcomes a long-standing challenge to incorporate nonnatural cofactors through de novo biosynthesis. By utilizing a ubiquitous laboratory strain, this process will facilitate spectroscopic studies and the development of enzymes for CoPPIX-mediated biocatalysis.

带有非天然或人工引入的金属中心的酶可以产生新的反应性，并使新的光谱和结构研究成为可能。对于金属有机辅因子，例如金属卟啉，不存在在体内构建和掺入新的天然辅因子类似物的通用方法。我们在这里报告一种常见的实验室菌株，大肠杆菌BL21(DE3)，在铁限制、富含钴的生长条件下生物合成钴原卟啉IX (CoPPIX)。_{在含有 CoCl 2}的补充基本培养基中，代谢产生的 CoPPIX 直接掺入多种血红素蛋白中，代替天然血红素b(FePPIX)。用这种新的天然钴卟啉辅因子成功表达了五种钴取代蛋白：肌红蛋白 H64V V68A、染料脱色过氧化物酶、醛肟脱水酶、细胞色素 P450 119 和过氧化氢酶。我们最终表明这些蛋白质包含 CoPPIX，其中 CoPPIX 占总卟啉含量的至少 95%。在天然金属配体是硫或氮的情况下，光谱参数与天然金属配体的保留一致。该方法在良率和易于实施方面对先前的方法进行了改进。值得注意的是，这种方法克服了通过从头生物合成掺入非天然辅助因子的长期挑战。通过利用无处不在的实验室菌株，