Integration of oxygen, light and tetrappyrole sensing in Rhodobacter sphaeroides

Tetrapyrroles are a family of compounds that contain four pyrrole rings. They are involved in many fundamental biological processes such as photoreception, electron transport, gas transport and as cofactors for enzymatic reactions. As regulators of protein activity, tetrapyrroles mediate cellular response to light, oxygen and nutrient levels in the surrounding environment. Among photosynthetic organisms, the heme and bacteriochlorophyll biosynthetic pathways share common intermediates from delta-aminolevulinic acid to protoporphyrin IX. Unbound heme and bacteriochlorophyll are both toxic thus the vast majority of these tetrapyrroles are sequestered within proteins. However, it is not clear how cells coordinate the synthesis of defined amounts of tetrapyrrole endproducts that utilize common intermediates with the synthesis of apo-proteins that bind tetrapyrroles.;In R. sphaeroides, heme and bacteriochlorophyll biosynthesis are co-regulated by the redox and light controlled PpsR-AppA system. PpsR is a DNA-binding transcription factor that recognizes conserved palindromes present in a number of tetrapyrrole and photosynthesis promoters. Under aerobic conditions, a pair of redox active Cys in PpsR undergo oxidation to stimulate binding of PpsR to target promoters to block transcription. PpsR is also regulated by the flavin-containing antirepressor AppA that responds to changes in both redox and light intensity. AppA inactivates PpsR by forming an inactive PpsR2-AppA complex under anaerobic dark conditions. Recently, it was reported that AppA also binds heme as a cofactor, though the role of heme bound to AppA remains ambiguous.;In this study, we present evidence that PpsR is a heme binding protein and that the redox active Cys424 present in the DNA binding domain of PpsR is critical for heme interaction. The binding of heme to PpsR was found to change its DNA-binding pattern, and to induce increased transcription of several PpsR regulated genes. We also further probe the roles of the BLUF, SCHIC and Cys-rich domains of AppA. We demonstrate that dark-adapted AppA binds heme better than light-excited AppA and that heme bound to the SCHIC domain significantly reduces the length of the BLUF photocycle. We also demonstrate that the interaction between heme and SCHIC domain is affected by the redox state of cysteine residues in the Cys-rich carboxyl terminal region. These results suggest light, redox and heme sensing are integrated in AppA, and that the involvement of redox and light in heme regulated AppA antirepression of PpsR is significantly more complicated than previously reported.