Refined Crystal Structure of Spinach Ferredoxin Reductase at 1.7 Å Resolution: Oxidized, Reduced and 2′-Phospho-5′-AMP Bound States

Abstract

The crystal structure of spinach ferredoxin-NADP+-oxidoreductase (FNR), determined by multiple isomorphous replacement at 2.6 Å resolution, has been refined at 1.7 Å resolution to anR-factor of 17.9%. The structure of FNR bound to the competitive inhibitor 2′-phospho-5′-AMP (P-AMP) has also been refined at 1.7 Å to anR-factor of 17.4% and dithionite-reduced/P-AMP-bound FNR has been refined at 2.0 Å to anR-factor of 14.9%. The P-AMP-bound structure was used to construct a model for the binding of NADP+. Over 200 solvation sites were included in each structure, and many of the best defined solvation sites stabilize buried turns. A bulk solvent correction obviated the need for a low-resolution data cutoff. An acidic side-chain likely to be responsible for the low pH requirement for cystallization has been identified. Three large networks of the hydrophobic side-chains help define the FNR structure. One of these contains a large cavity far from the active site, which coincides with the lone site of sequence heterogeneity in FNR, and may provide a site for membrane attachment. The reduced structure shows that Ser96 moves toward atom N-5 of FAD and a water molecule moves toward atom N-1 of FAD, while the flavin moiety remains planar. Possible sources of a proton that must be picked up upon reduction are discussed. © 1995 Academic Press Limited.