Structural and mechanistic insights into the electron flow through protein for cytochrome c-tethering copper nitrite reductase.

Copper-containing nitrite reductases (CuNiRs), which catalyse the reversible one-electron reduction of nitrite to nitric oxide, are members of a large family of multi-copper enzymes that require an interprotein electron transfer (ET) reaction with redox partner proteins. Here, we show that the naturally fused type of CuNiR tethering a cytochrome c (Cyt c) at the C-terminus folds as a unique trimeric domain-swapped structure and has a self-sufficient electron flow system. The C-terminal Cyt c domain is located at the surface of the type 1 copper (T1Cu) site in the N-terminal CuNiR domain from the adjacent subunit, the heme-to-Cu distance (10.6 Å) of which is comparable to the transient ET complex of normal CuNiR with Cyt c. The structural aspects for the domain-domain interface and the ET kinetics indicate that the Cyt c-CuNiR domain interaction should be highly transient. The further electrochemical analysis of the interprotein ET reaction with a cognate redox partner protein suggested that an electron is directly transferred from the partner to the T1Cu. Structural and mechanistic comparisons of Cyt c-CuNiR with another cupredoxin-tethering CuNiR highlight the behaviours of extra domains on the fusion types of CuNiRs required for ET through proteins.