Electron transfer to nitrite reductase of Rhodobacter sphaeroides 2.4.3: examination of cytochromes c 2 and c Y

Abstract

The role of cytochromec2, encoded bycycA, and cytochromecY, encoded bycycY, in electron transfer to the nitrite reductase ofRhodobacter sphaeroides2.4.3 was investigated using bothin vivoandin vitroapproaches. BothcycAandcycYwere isolated, sequenced and insertionally inactivated in strain 2.4.3. Deletion of either gene alone had no apparent effect on the ability ofR. sphaeroidesto reduce nitrite. In acycA–cycYdouble mutant, nitrite reduction was largely inhibited. However, the expression of the nitrite reductase genenirKfrom a heterologous promoter substantially restored nitrite reductase activity in the double mutant. Using purified protein, a turnover number of 5 s−1was observed for the oxidation of cytochromec2by nitrite reductase. In contrast, oxidation ofcYonly resulted in a turnover of ∼0·1 s−1. The turnover experiments indicate thatc2is a major electron donor to nitrite reductase butcYis probably not. Taken together, these results suggest that there is likely an unidentified electron donor, in addition toc2, that transfers electrons to nitrite reductase, and that the decreased nitrite reductase activity observed in thecycA–cycYdouble mutant probably results from a change innirKexpression.