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Electron transfer to nitrite reductase of Rhodobacter sphaeroides 2.4.3: examination of cytochromes c₂ and c_Y

William P. Laratta

Department of Microbiology, Wing Hall, Cornell University, Ithaca, NY 14850-8101, USA

Correspondence
James P. Shapleigh
jps2{at}cornell.edu

The role of cytochrome c₂, encoded by cycA, and cytochrome c_Y, encoded by cycY, in electron transfer to the nitrite reductase of Rhodobacter sphaeroides 2.4.3 was investigated using both in vivo and in vitro approaches. Both cycA and cycY were isolated, sequenced and insertionally inactivated in strain 2.4.3. Deletion of either gene alone had no apparent effect on the ability of R. sphaeroides to reduce nitrite. In a cycA–cycY double mutant, nitrite reduction was largely inhibited. However, the expression of the nitrite reductase gene nirK from a heterologous promoter substantially restored nitrite reductase activity in the double mutant. Using purified protein, a turnover number of 5 s^–1 was observed for the oxidation of cytochrome c₂ by nitrite reductase. In contrast, oxidation of c_Y only resulted in a turnover of 0·1 s^–1. The turnover experiments indicate that c₂ is a major electron donor to nitrite reductase but c_Y is probably not. Taken together, these results suggest that there is likely an unidentified electron donor, in addition to c₂, that transfers electrons to nitrite reductase, and that the decreased nitrite reductase activity observed in the cycA–cycY double mutant probably results from a change in nirK expression.

Present address: Agave Biosystems, 401 East State Street, Ithaca, NY 14850, USA.