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As part of a series of comparisons of the structures of the three oxidation states of flavodoxin from Clostridium MP, phases for the semiquinono form were determined to 2.0 A resolution by isomorphous replacement (m> = 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 versus maps with Fourier coefficients of the form (2|Fo|- |Fc|) exp (i[alpha]c). To minimize bias in the fitting, groups of atoms were systematically omitted from the structure factors used in computation of the (2|Fo - |Fc|) 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 [deg] for the semiquinone structure and ~0 [deg] in oxidized flavodoxin. The intensity changes resulting from the oxidized agsemiquinone conversion (RI = 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 is