Genetically engineered strains of Escherichia coli and Pseudomonas aeruginosa were prepared harboring the gene cluster nirFDLGH from Pseudomonas stutzeri substrain ZoBell on a high copy plasmid. These genes have been previously implicated as being essential for the biosynthesis of heme d(1), the prosthetic group of dissimilatory nitrite reductases in anaerobic, denitryfying bacteria. Tetrapyrroles detectable at steady-state levels were identified from both organisms, and cell-free extracts from each were also used to transform uroporphyrinogen in vitro. E. coli does not naturally produce d(1), and the engineered strain failed to produce d(1) or any tetrapyrrole foreign to E. coli. Therefore, while nirFDLGHmay be necessary for d(1) biosynthesis, it is not sufficient. In the denitrifier P. aeruginosa, the results were more positive. The presence of the plasmid led to increased levels of d(1). In addition, a previously unidentified tetrapyrrole was detected. This compound was characterized by visible absorption spectroscopy, infrared spectroscopy, X-ray photoelectron spectroscopy, mass spectrometry, and NMR, and a tentative structure was proposed for this compound. The tetrapyrrole has structural features similar to sirohydrochlorin (as precorrin-2 or sirotetrahydrochlorin, a known intermediate of d(1)) and d(1) itself. The most unusual substituents are epoxide and sulfoxide moieties. When this tetrapyrrole was treated with strong mineral acid and heat, it was converted into natural d(1).