Crystallographic location of two Zn²⁺ binding sites in the avian cytochrome bc₁ complex

Lawrence Berkeley National Laboratorya,

The Graduate Group of Biophysicsb, University of California, Berkeley,

and Stanford Synchrotron Radiation Laboratory^c

Figure 1. Inhibition of the ubiquinol:cytochrome c reductase activity of purified chicken bc₁ complex by ZnCl₂. Electron transfer from the decyl analog of ubiquinone to horse heart cytochrome c was assayed as described in materials and methods. Open symbols: Inhibition by ZnCl₂ alone. Closed symbol: Inhibition by 2 mM myxothiazol as well as 200 mM ZnCl₂.

Figure 2. Stereo view of Zn01 in its binding site. The site of Zn01 (cyan sphere near center) is in the interface between cytochrome b (salmon), cytochrome c₁ (blue), and the ISP (green) The ef linker of cytochrome b, which provides 3 of the 4 potential ligands for Zn01, is highlighted with a darker shade. The yellow and red ball-and-stick model to the right and below Zn01 is stigmatellin from the superimposed 3BCC structure (see legend to Figure 3). Glu272 is visible between stigmatellin and the low potential heme of cytochrome b. The green sphere to the left and behind Zn01, shown bonded to R81 of cytochrome b, is the density mentioned in the text that is present in all crystals, independent of ZnCl₂ treatment, and is modeled as a Cl^- ion. Zn02 is also visible, partially obscured by the horizontal ef helix. M125 is shown as a ball-and-stick model below and to the left of Zn02. The figure was made with Molscript [21] and Raster3D [22].

Figure 3. View of Zn02 binding in the hydrophobic channel between the Q_o site and the bulk lipid phase. Stereo view of the cytochrome b backbone (red Ca trace) with the residues around the channel portrayed as wireframe (above) and space-filling (below) models, viewed from the position of the second cytochrome b monomer. Stigmatellin is the thick yellow and red stick model, with the hydrophobic tail pointed toward the viewer. (The orientation is related to that of Figure 2 by approximately 180 rotation about the vertical axis) When viewed in stereo the residues depicted are seen to form a channel around the hydrophobic tail of stigmatellin. The cyan sphere in the middle of the channel, through which the stigmatellin passes, represents the position of Zn02. The green sphere in the wall of the channel in the space-filling model is Sd of MET125, the protein atom most closely associated with the zinc. The stigmatellin coordinates are from an updated version of the coordinates deposited as 3BCC. The zinc is from the current structure. Both coordinate sets were transformed to a standard reference cell in order to superimpose structures from slightly different cells. The figure was made using the graphics program O [23]

Figure 4. Alignment of vertebrate bc₁ complex subunit one with the inverse zinc-binding motif of b-MPP. Residues making up the motif, (HXXEH-Xn-E), are boxed. The histidine which is mutated to Y in core 1 protein is indicated by a vertical mark above the first row. In some cases, notably Neurospora and potato, b-MPP is subunit 1 of the bc₁ complex.