The 2.6 A resolution structure of Rhodobacter capsulatus bacterioferritin with metal-free dinuclear site and heme iron in a crystallographic 'special position'

Abstract

Bacterioferritin from Rhodobacter capsulatus was crystallized and its structure was solved at 2.6 A resolution. This first structure of a bacterioferritin from a photosynthetic organism is a spherical particle of 24 subunits displaying 432 point-group symmetry like ferritin and bacterioferritin from Escherichia coli. Crystallized in the I422 space group, its structural analysis reveals for the first time the non-symmetric heme molecule located on a twofold crystallographic symmetry axis. Other hemes of the protomer are situated on twofold noncrystallographic axes. Apparently, both types of sites bind heme in two orientations, leading to an average structure consisting of a symmetric 50:50 mixture, thus satisfying the crystallographic and noncrystallographic symmetry of the crystal. Five water molecules are situated close to the heme, which is bound in a hydrophobic pocket and axially coordinated by two crystallographic or noncrystallographically related methionine residues. Its ferroxidase center, in which Fe(II) is oxidized to Fe(III), is empty or fractionally occupied by a metal ion. Two positions are observed for the coordinating Glu18 side chain instead of one in the E. coli enzyme in which the site is occupied. This result suggests that the orientation of the Glu18 side chain could be constrained by this interaction.

Figure 1

stereoview of the Rhodobacter capsulatus bacterioferritin subunits of the asymmetric unit schematized as a ribbon. The heme group is represented in ball-and-stick. Monomers related by a noncrystallographic 2-fold axis are colored in red (A) and green (B). The subunit (C) binding to the heme situated on the 2-fold crystallographic axis is colored in blue. (a) Trimeric structure including one heme with its enclosing noncrystallographic dimer and one heme on a crystallographic axis (at occupancy 0.5) with one monomer of its enclosing dimer. (b) Trimeric structure with proper 3-fold symmetry, including three heme moieties at 0.5 occupancy. The Met52 and heme are represented in ball-and-stick. The drawing was generated using Molscript (19) and Raster3D (20).

Figure 2

stereoview of the Bfr protomer from R. capsulatus along the 3-fold channel (a), along the 4-fold channel situated on the four-fold crystallographic axis (b) and along the 2-fold axis (c). The drawing was generated using Molscript (19).

Figure 3

stereoview of the 4-fold channel along the 4-fold crystallographic axis. The Leu151 residues situated in the channel are displayed in ball-and-stick. The drawing was generated using Molscript (19) and Raster3D (20).

Figure 4

stereoview of the 3-fold channel. Arg105, Lys117, Asn118 and Glu119 are represented in ball-and-stick. The drawing was generated using Molscript (19) and rendered using Raster3D (20).

Figure 5

stereoview of the two positions of the Glu18 side chain in a Fo-Fc electron density map (a) contoured at +3.5 σ (green) and −3.5 σ (red) and in a 3Fo-2Fc omit map (b) contoured at 1.2 σ and calculated at 2.6 Å resolution. The Ser17, Glu18 and Leu19 side chains are displayed in ball-and-stick. The two positions for the carboxylate group are displayed in different colors. The drawing was realized using Molscript (19) and O (16) and rendered using Raster3D (20).

Figure 6

stereoview of the two positions of the heme situated on the 2-fold crystallographic axis (a) and on the 2-fold noncrystallographic axis (b) in a 3Fo-2Fc omit map calculated at 2.6 Å resolution and contoured at 1.2 □. The two orientations of the heme are showed in green and red colors. For (a) the map was calculated from data reduced in the space group I4 to avoid imposing symmetry, and in both figures the heme was omitted from the phasing model. The drawing was generated using Molscript (19) and O (16) and rendered using Raster3D (20).