Cytochromes c ₅₅₀ , c ₅₅₂ , and c ₁ in the Electron Transport Network of Paracoccus denitrificans : Redundant or Subtly Different in Function?

Abstract

Paracoccus denitrificans strains with mutations in the genes encoding the cytochrome c 550 , c 552 , or c 1 and in combinations of these genes were constructed, and their growth characteristics were determined. Each mutant was able to grow heterotrophically with succinate as the carbon and free-energy source, although their specific growth rates and maximum cell numbers fell variably behind those of the wild type. Maximum cell numbers and rates of growth were also reduced when these strains were grown with methylamine as the sole free-energy source, with the triple cytochrome c mutant failing to grow on this substrate. Under anaerobic conditions in the presence of nitrate, none of the mutant strains lacking the cytochrome bc 1 complex reduced nitrite, which is cytotoxic and accumulated in the medium. The cytochrome c 550 -deficient mutant did denitrify provided copper was present. The cytochrome c 552 mutation had no apparent effect on the denitrifying potential of the mutant cells. The studies show that the cytochromes c have multiple tasks in electron transfer. The cytochrome bc 1 complex is the electron acceptor of the Q-pool and of amicyanin. It is also the electron donor to cytochromes c 550 and c 552 and to the cbb 3 -type oxidase. Cytochrome c 552 is an electron acceptor both of the cytochrome bc 1 complex and of amicyanin, as well as a dedicated electron donor to the aa 3 -type oxidase. Cytochrome c 550 can accept electrons from the cytochrome bc 1 complex and from amicyanin, whereas it is also the electron donor to both cytochrome c oxidases and to at least the nitrite reductase during denitrification. Deletion of the c-type cytochromes also affected the concentrations of remaining cytochromes c, suggesting that the organism is plastic in that it adjusts its infrastructure in response to signals derived from changed electron transfer routes.

A Mutant of Paracoccus denitrificans with Disrupted Genes Coding for Cytochrome c 550 and Pseudoazurin Establishes These Two Proteins as the In Vivo Electron Donors to Cytochrome cd 1 Nitrite Reductase

“…The implication is again that only a copper-containing protein can substitute for cytochrome c 550 in nitrite reduction during denitrification. As might be predicted on this basis, a double deletion mutant with deletion of both cytochrome c 550 and cytochrome c 552 also requires copper in the medium for anaerobic growth on succinate plus nitrate (33). Cytochrome c 552 is expressed constitutively, and its globular heme-containing domain has a structure very similar to that of cytochrome c 550, although it possesses an acidic patch and overall has a less positive charge (18).…”

“…presence of nitrite at millimolar levels disrupts the pattern of growth away from the ideal behavior (33). In the wild-type and single mutant cells nitrite formation correlated with the early stage of growth following any lag phase, but why different strain-dependent nitrite concentrations were reached (Fig.…”

“…Growth characteristics. It was not thought that aerobic growth on a heterotrophic carbon and free energy source should require either cytochrome c 550 or pseudoazurin, because cytochrome c 552 is a protein that accepts electrons from the cytochrome bc 1 complex and delivers them to the cytochrome aa 3 oxidase (33), while cytochrome ba 3 , which is a quinol oxidase, functions independent of either pseudoazurin or cytochrome c 550 (15,33). This expectation was confirmed by the finding that aerobic growth with succinate as a carbon and free energy source was not affected in any of the mutants.…”

“…However, another possible interpretation has been put forward on the basis of the observations made with diethyldithiocarbamate (24). A further indication of the importance of a copper protein in the absence of cytochrome c 550 came from the observation that the mutant grew anaerobically much more poorly in growth media with low copper contents than did the wild-type organism (33,45). Furthermore, the nitrite reductase exhibits activity in vitro with pseudoazurin as an electron donor (29).…”

“…This is because a mutant of P. denitrificans that lacks the gene (cycA) encoding cytochrome c 550 is still able to reduce nitrite; indeed, no respiratory activity of P. denitrificans is lost as a result of interruption of the cycA gene (44). The role of the cytochrome bc 1 complex in electron delivery to cytochrome cd 1 has been established through studies with both inhibitors (2) and mutants specifically deficient in this complex (33). In the case of nitrite reduction, it was proposed by Moir and Ferguson (30) that the absence of cytochrome c 550 was compensated for by another protein that was able to act as an electron donor to cytochrome cd 1 .…”