Abstract
Denitrification is a microbial pathway that constitutes an important part of the nitrogen cycle on earth. Denitrifying organisms use nitrate as a terminal electron acceptor and reduce it stepwise to nitrogen gas, a process that produces the toxic nitric oxide (NO) molecule as an intermediate. In this work, we have investigated the possible functional interaction between the enzyme that produces NO; the cd1 nitrite reductase (cd1NiR) and the enzyme that reduces NO; the c-type nitric oxide reductase (cNOR), from the model soil bacterium P. denitrificans. Such an interaction was observed previously between purified components from P. aeruginosa and could help channeling the NO (directly from the site of formation to the side of reduction), in order to protect the cell from this toxic intermediate. We find that electron donation to cNOR is inhibited in the presence of cd1NiR, presumably because cd1NiR binds cNOR at the same location as the electron donor. We further find that the presence of cNOR influences the dimerization of cd1NiR. Overall, although we find no evidence for a high-affinity, constant interaction between the two enzymes, our data supports transient interactions between cd1NiR and cNOR that influence enzymatic properties of cNOR and oligomerization properties of cd1NiR. We speculate that this could be of particular importance in vivo during metabolic switches between aerobic and denitrifying conditions.
Highlights
Denitrification is a microbial pathway that constitutes an important part of the nitrogen cycle on earth
The enzyme catalyzing the reduction of nitrite to nitric oxide (NO2− + e− + 2 H+ →NO + H2O) in P. denitrificans is cytochrome cd[1] nitrite reductase, a soluble protein located in the periplasm
The aim of this work was to determine whether the P. denitrificans cd1 nitrite reductase (cd1NiR) and c-type nitric oxide reductase (cNOR) form a molecular complex in vivo and/or in vitro and to study potential functional interactions in vitro
Summary
Denitrification is a microbial pathway that constitutes an important part of the nitrogen cycle on earth. We have investigated the possible functional interaction between the enzyme that produces NO; the cd[1] nitrite reductase (cd1NiR) and the enzyme that reduces NO; the c-type nitric oxide reductase (cNOR), from the model soil bacterium P. denitrificans Such an interaction was observed previously between purified components from P. aeruginosa and could help channeling the NO (directly from the site of formation to the side of reduction), in order to protect the cell from this toxic intermediate. The well-characterized cd1NiRs from P. pantotrophus and Pseudomonas (Ps) aeruginosa (see e.g.9 and4) have many properties in common including a similar overall fold especially in the larger, catalytic d1 domain They use similar electron donors; a soluble c cytochrome or a blue copper protein. There are striking differences, such as the ‘domain swapping’ that occurs only in the Ps. aeruginosa cd1NiR dimer, where the N-terminal arm (in the cyt. c domain) of one monomer crosses over to interact with the d1 domain of the second monomer
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