Structural Studies on Corn Nitrate Reductase: Refined Structure of the CytochromebReductase Fragment at 2.5 Å, its ADP Complex and an Active-site Mutant and Modeling of the CytochromebDomain

Abstract

The refined crystal structures of the recombinant cytochromebreductase fragment of corn (Zea mays) nitrate reductase, its ADP complex and the active-site mutant Cys242Ser are reported here. The native structure has been refined at 2.5 Å resolution to a crystallographicR-factor of 18.7% with root-mean-square (r.m.s.) deviations from standard bond lengths and angles of 0.013 Å and 2.0°. The diffraction pattern of the crystals is highly anisotropic and correction of this effect lowered the crystallographicR-factor by 5% during the refinement. The structure of the enzyme co-crystallized with ADP has been solved at 2.7 Å resolution and refined to anR-factor of 18.6% with r.m.s. deviations from standard bond lengths and angles of 0.014 Å and 2.1°. It revealed the binding site of the ADP moiety of the NADH cofactor, which is the electron donor for nitrate reduction. Based on this structure, a model of NADH at the active site of the enzyme was built and the implications for electron transfer from NADH to the flavin cofactor are discussed. The crystal structure of an active-site mutant enzyme, Cys242Ser, has been solved by difference Fourier synthesis and refined to anR-factor of 19.0% to 3.0 Å resolution with standard deviations of bond lengths and angles of 0.017 Å and 2.5°. This structure analysis suggests that the observed decrease in catalytic activity of this mutant might be due to misalignment of the nicotinamide ring in its binding site. A model of the heme-containing domain of nitrate reductase has been built based on the X-ray structure of bovine cytochromeb5and has been docked with the cytochromebreductase fragment of nitrate reductase. The model of the complex contains six salt-bridges at the domain-domain interface and a hydrophobic core. In this model, His48, an invariant residue in the cytochromebreductase family, forms an interaction with the propionic acid group of the D-ring of the heme cofactor. This group is in contact with the C-8 methyl group of the flavin ring. Residues that might influence the redox potential of the flavin cofactor are proposed and their possible role in electron transfer is discussed.