Temporal expression of Mycobacterium smegmatis respiratory terminal oxidases.

Terminal oxidases provide the final step in aerobic respiration by reducing oxygen. The mycobacteria possess two terminal oxidases: a cytochrome c aa3 type and a quinol bd type. We previously isolated a bd-type oxidase knockout mutant of Mycobacterium smegmatis that allowed for functional analysis of the aa3 type without the contribution of bd-type activity. Growth of M. smegmatis LR222 and JAM1 (LR222bd::kan) was monitored and the cytochrome content at different time points examined. No difference in aerobic growth was observed between M. smegmatis LR222 and JAM1. Membranes were obtained from these cultures and the oxidase concentrations were calculated from their spectrum. Although the mutant was producing only one oxidase type, this oxidase did not reach wild-type levels of expression, suggesting an additional mechanism for energizing the membrane. Moreover, the concentration of both oxidases in the wild-type strain dropped when cultures entered stationary phase, which was not the case for the aa3-type oxidase of the mutant strain. This oxidase remained at a constant concentration post mid-log phase. RNase protection assays also demonstrated late growth phase dependent message expression of the bd oxidase and that the subunits I and II genes were cotranscribed as an operon.

Evidence for a cytochrome bcc-aa3 interaction in the respiratory chain of Mycobacterium smegmatis.

Spectroscopic analysis of membranes isolated from Mycobacterium smegmatis, along with analysis of its genome, indicates that the cytochrome c branch of its respiratory pathway consists of a modified bc1 complex that contains two cytochromes c in its c1 subunit, similar to other acid-fast bacteria, and an aa3-type cytochrome c oxidase. A functional association of the cytochrome bcc and aa3 complexes was indicated by the findings that levels of detergent sufficient to completely disrupt isolated membranes failed to inhibit quinol-driven O2 reduction, but known inhibitors of the bc1 complex did inhibit quinol-driven O2 reduction. The gene for subunit II of the aa3-type oxidase indicates the presence of additional charged residues in a predicted extramembrane domain, which could mediate an intercomplex association. However, high concentrations of monovalent salts had no effect on O2 reduction, suggesting that ionic interactions between extramembrane domains do not play the major role in stabilizing the bcc-aa3 interaction. Divalent cations did inhibit electron transfer, likely by distorting the electron-transfer interface between cytochrome c1 and subunit II. Soluble cytochrome c cannot donate electrons to the aa3-type oxidase, even though key cytochrome c-binding residues are conserved, probably because the additional residues of subunit II prevent the binding of soluble cytochrome c. The results indicate that hydrophobic interactions are the primary forces maintaining the bcc-aa3 interaction, but ionic interactions may assist in aligning the two complexes for efficient electron transfer.