Structure of a protein able to trap selenium
A) Aim of the study
The periplasmic nitrate reductase of Rhodobacter sphaeroides (Nap) is a dimeric metalloprotein that catalyses the reduction of nitrates to nitrite, the first step in the denitrification process (respiration of nitrates). It consists of 1) a NapA subunit of 90 kDa binding a 4Fer-4S cluster and a molybdenum cofactor (MGD), and 2) a NapB subunit (18 kDa) of the cytochrome type able to transfer electrons towards the molybdenum centre where the nitrate reduction reaction takes place. We have shown that the enzyme can also reduce metal oxides such as seleniates and tellurites, although its reductive activity for these compounds remains low (Sabaty et al., 2001). However, its structure suggests that these activities could be enhanced.
B) Resolution of the crystallographic structure
The NapA-NapB complex was cloned and expressed in Rhodobacter sphaeroides. In an intensive study, combined purification and crystallisation yielded 3 different crystalline forms (Pignol et al., 2001). We recorded the diffraction spectra on line ID14 of the ESRF (Grenoble). At CEA Cadarache we used these data to resolve the structure of the complex by a combination of methods of molecular replacement and averaging. The structure was refined at 3.1 Å resolution.
C) Description of the structure
The catalytic subunit NapA contains a molybdenum atom (MGD cofactor) located far inside a crevice corresponding to the substrate binding site. The NapB subunit presents a stretched conformation and lies throughout its length on the NapA subunit. The interaction surface area between the two subunits is about 6000 Å3, a very high value, which explains the very high affinity measured between the two partners. The structure gives us the precise topology of the metal cofactors. The electrons necessary for the reduction of the nitrates are transferred first from haem I to haem II of NapB, then to the cluster (4Fe-4S) and finally to the molybdenum of the NapA. The distances between the metal cofactors are compatible with a direct electron transfer.
Our study also enabled us for the first time to elucidate the structure of a subunit presenting a c-type haem associated with a subunit possessing a 4Fe-4S cluster. The analysis of the interactions between the two metal cofactors is described in detail in our paper.
D) Enzyme studies
To characterise the role of the two subunits we cloned and overexpressed NapA and NapB separately. The enzyme kinetics were measured on NapA, NapAB and ranging NapA/NapB ratios. The results showed that:
- NapB is an activator. The electron transfer, the rate limiting step in the reaction, is accelerated fivefold by the cytochrome subunit.
- NapA works by a ‘ping-pong' mechanism. There is first an electron transfer from haem I to the active centre and reduction of the molybdenum. In a second step, binding of the substrate (nitrate or seleniate) and oxidation of the molybdenum takes place, accompanied by reduction of the substrate.
E) Spectroscopic study
From the purified proteins NapA, NapB and the complex NapAB, we measured the redox potentials of the different cofactors in their free and complexed forms using methods of visible-range and EPR spectroscopy.
These results enabled us to assign each potential to its corresponding cofactor. They also show variability between free and complexed forms. These variations correspond to cofactors located at the interface between the subunits (haem II and cluster Fer-S) and point to structural plasticity. These modifications in potentials measured during the formation of the complex produce a highly favourable electron transfer between each cofactor, which is not the case for the potentials of the free subunits. This last observation is linked to the activating effect of the NapB.
This work was presented in a guest talk at the Gordon Research Conference on Molybdenum and Tungsten Enzymes held on 29 June to 4 July at Meriden NH, USA and is published in the November 2003 issue of Nat. Struct. Biol. (Arnoux P., Sabaty M., Alric J., Frangioni B., Guigliarelli B., Adriano J.M. and Pignol D.)