Oxygen-Mediated Regulation of Porphobilinogen Formation in Rhodobacter capsulatus

It has been almost a half-century since the work of Cohen-Bazire et al. (13) demonstrated that oxygen regulates bacteriochlorophyll synthesis in the purple nonsulfur photosynthetic bacteria. However, the mechanism by which oxygen regulates this pathway has been difficult to elucidate. For Rhodobacter capsulatus, it has been shown that a bchH mutant accumulates much more protoporphyrin IX when grown under low oxygen tension than under high oxygen tension (6), indicating that oxygen regulates some step in the common tetrapyrrole pathway (Fig. ​(Fig.1).1). Rebeiz and Lascelles (39) proposed that oxygen inhibits the chelation of magnesium into the protoporphyrin ring, the first step in the branch leading to bacteriochlorophyll. The accumulating protoporphyrin IX would be diverted to heme, which would feedback inhibit aminolevulinate synthase. Inhibition of this enzyme would reduce carbon-flow over the common tetrapyrrole pathway. FIG. 1. The common tetrapyrrole pathway in R. capsulatus. Dashed lines denote multistep pathways. This theory was tested in R. capsulatus by growing a bchH mutant (AJB456) under high and low oxygen concentrations in the presence of exogenous aminolevulinate (5). Surprisingly, protoporphyrin IX accumulation was still regulated by oxygen. However, when exogenous porphobilinogen, the second intermediate in the pathway, was added to a culture of the bchH mutant, protoporphyrin IX accumulation was no longer regulated by oxygen (5). This observation indicated that the major oxygen-regulated control point in the common tetrapyrrole pathway is not the synthesis of aminolevulinate. Rather, it appears that oxygen regulates the second step in the pathway, the conversion of aminolevulinate to porphobilinogen. This is reminiscent of the situation in Bradyrhizobium japonicum, where transcription of the hemB gene is regulated by oxygen (10). The lack of a hemC mutant has made it difficult to confirm and extend these results. The most straightforward way to determine whether oxygen regulates porphobilinogen formation in R. capsulatus would be to measure porphobilinogen levels in a hemC mutant. Such a mutant would lack porpobilinogen deaminase and would therefore accumulate porphobilinogen. In this study, the construction and characterization of a hemC mutant is detailed. Using this mutant, it was determined that the intracellular porphobilinogen level is regulated by oxygen tension. Since the situation in R. capsulatus appears to be similar to that of B. japonicum (10), transcriptional regulation of the hemB gene was investigated. We report here that hemB transcription is not regulated in response to oxygen tension nor is the activity of porphobilinogen synthase feedback inhibited by either hemin or protoporphyrin IX. We did observe, however, that intracellular hemin levels appear to influence carbon flow over the common tetrapyrrole pathway. Additionally, R. capsulatus has a novel enzyme, aminolevulinate dehydrogenase, which reduces aminolevulinate to aminohydroxyvalerate. The regulation of porphobilinogen levels appears to be mediated by a combination of feedback inhibition of aminolevulinate synthase by hemin and by diverting aminolevulinate from the common tetrapyrrole pathway.