Structure of the semiquinone form of flavodoxin from Clostridum MP. Extension of 1.8 A resolution and some comparisons with the oxidized state.

As part of a series of comparisons of the structures of the three oxidation states of flavodoxin from Clostridium MP, phases for the semiquinonc form were determined to 2.0 A resolution by isomorphous replacement ( = 0.725). Subsequently, the structure was refined at 1.8 A resolution by a combination of difference Fourier, real space and reciprocal space methods. After refining to an R of 0.194, we explored the conformation of the FMN binding site by real space refinement verau6 maps with Fourier coefficients of the form (2) F,I 1 P,I ) exp (in,). To minimize bias in the fitting, groups of atoms were systematically omitted from the structure factors used in computat.ion of the (212”,1 IF,\) maps. One-electron reduction of oxidized flavodoxin is accompanied by several changes at the FMN binding site: the conformation of residues in the reverse bend formed by Met56-Gly57-Asp58-Glu59 differs in the crystal structures of the oxidized and semiquinone species; further, backbone atoms in residues 55 and 89 shift by more than 0.5 A and the indole ring of Trp90 undergoes a significant displacement. The orientation of the peptide unit connecting Gly57 and Asp58 is consistent with the presence of a hydrogen bond between the carbonyl oxygen of Gly57 and the flavin N(5) in flavodoxin semiquinone. No equivalent bond is found in oxidized flavodoxin. In both the oxidized and semiquinone species of clostridial flavodoxin, the isoalloxazine ring is essentially planar : the bending angles about N(5)-N( 10) are -2.5” for the semiquinone structure and ~0’ in oxidized flavodoxin. The intensity changes resulting from t,he oxidized,semiquinone conversion (K, m= 0.33) arise in part from changes in molecular packing. Intermolecular contacts, including neighbors of the prosthetic group, are altered in the repacking. Maps or models of the two oxidation states can be brought into approximate coincidence by a rigid body motion. The required transformation, determined for the isomorpllous replacement maps by the method of Cox (1967), is equivalent to a screw motion with a rotation of 1.18” and a translation of -0.34 A. The molecular structures of oxidized and semiquinone flavodoxins have been compared after superposition of models with idealized co-ordinates and discrepancy indices R,, = 0.213 and R,, = 0.200. The root-mean-square distance between 523 backbone atoms (excluding sequences 56 to 59 and 89 to 91) is O-308 A.