Cytochrome d expression and regulation pattern in free-living Rhizobium phaseoli

The respiratory system of Rhizobium phaseoli CFN42 in free-living cultures was studied. Cytochromes b, c, o and aa ₃ were found in fast growing cells cultured under forced aeration. Stationary aerobic cells, and semianaerobically grown cells showed decreased levels of cytochromes c, aa ₃ and o, concomitant with a significant increase of b type cytochromes and the synthesis of a new cytochrome, tentatively identified as cytochrome d. Cell membranes with the highest content of cytochrome d (semianaerobically grown cells) showed the highest respiratory activities with NADH, succinate, malate or ascorbate-TMPD (N,N,N,N-tetramethyl p-phenylendiamine). In the presence of either of the above electron donors, cytochrome d was clearly reduced. NADH dependent respiration in membranes of fast growing cells (no cytochrome d detected) was abolished by 25 M KCN. This inhibitor concentration caused only 15–20% inhibition in membranes of semianaerobically grown cells (cyt d present). Moreover, in the presence of 1–5 mM KCN, the oxidation of cyt d and a b type cytochromes was spectrally detected. It is suggested that cyt d is a functional cytochrome in the respiratory system of free-living Rhizobia, probably acting as terminal oxidase.