Complex oxidation of apocytochromes during bacterial cytochrome maturation.

-Type cytochromes (cyts ) are proteins that contain covalently bound heme, and thus require post-translational modification for activity, a process carried out by cyt maturation system (Ccm) in many Gram-negative bacteria. It has been established that during cyt maturation (CCM), two cysteine thiols of the heme binding motif (CXXCH) within apocyts are first oxidized largely by DsbA to form a disulfide bond, which is later reduced through a thio-reductive pathway involving DsbD. However, the physiological impacts of DsbA proteins on CCM in fact vary significantly among bacteria. In this work, we used Gram-negative, cyt -rich as the research model to clarify roles of DsbA proteins in CCM. We show that in terms of oxidation of apocyts , DsbA proteins are an important but not critical factor, and strikingly, oxygen is not either. By exploiting the DsbD-independent pathway, we identify DsbA1, and DsbA2, and DsbA3 as oxidants contributing to oxidation of apocyts and reductants such as cysteine as an effective antagonist against DsbA-independent oxidation. We further show that DsbB proteins are partially responsible for re-oxidization of reduced DsbA proteins. Overall, our results indicate that the DsbA-DsbB redox pair has a limited role in CCM, challenging the established notion that it is the main oxidant for apocyts DsbA is a powerful oxidase that functions in the bacterial periplasm to introduce disulfide bonds in many proteins, including apocytochromes It has been well established that DsbA, although not essential, plays a primary role in cytochrome maturation based on studies in bacteria hosting several cyts Here, with cyt -rich as a research model, we show that this is not always the case. Moreover, we demonstrate that DsbB is not essential for cytochrome maturation either. These results underscore the need to identify oxidants other than DsbA/DsbB that are crucial in oxidation of apocyts in bacteria.