The cytochrome (cyt) bc1 complex is one of the major contributors of the transmembrane electrochemical proton gradient used for ATP synthesis. The mechanism of redox coupled H+ translocation by the cyt bc1 complex involves two catalytic sites facing the two opposite sides of the energy transducing membrane: Qo , at which quinol oxidation is coupled to proton release and Qi , where quinone reduction is coupled to H+ uptake. Zn2+ is a well established inhibitor of the bovine mitochondrial bc1 complex (1), in which it competes with proton binding (2). In bacterial cyt bc1 complexes Zn2+ has been shown to decelerate electron transfer and transmembrane voltage generation (3). It has been proposed that Zn2+ binds close to the Q0 site, blocking the proton release channel(s). Two Zn2+ binding sites have been located in the avian cyt bc1 complex by X-ray diffraction: one of them, located in a hydrophilic area between the cyt b and c1, might interfere with the egress of H+ (4). To determine the coordination geometry of the bound metal we performed Zn K-edge X-ray Absorption Spectroscopy (XAS) measurements on Zn incubated samples of avian, bovine and bacterial (from Rhodobacter capsulatus (5)) cyt bc1 complexes. Zn stoichiometries were measured by ICP-emission spectroscopy, assuming 5 iron atoms per cyt bc1 complex. Samples were incubated with sub-stoichiometric amounts of Zn to maximize the occupancy of the high affinity site(s) minimizing that of lower affinity site(s). Preliminary analysis of both the avian and the bovine complexes indicates one histidine nitrogen and two oxygen atoms (at 2.00 Å distance) plus one nitrogen or oxygen atom as metal ligands. This agrees with the crystallographic binding site of the avian complex formed by His121, Asp253 and Glu255 (4). First-shell analysis in the bacterial complex suggests two nitrogen and two oxygen atoms (at 2.20 Å) plus one oxygen (or nitrogen) atom as ligands. In order to better define the local structure and possibly locate the cluster of binding residues a multi-shell, multiple scattering analysis is in progress.

X-ray absorption studies of Zn2+ binding sites in bacterial, avian and bovine cytochrome bc(1) complexes

GIACHINI, LISA;FRANCIA, FRANCESCO;BOSCHERINI, FEDERICO;VENTUROLI, GIOVANNI
2006

Abstract

The cytochrome (cyt) bc1 complex is one of the major contributors of the transmembrane electrochemical proton gradient used for ATP synthesis. The mechanism of redox coupled H+ translocation by the cyt bc1 complex involves two catalytic sites facing the two opposite sides of the energy transducing membrane: Qo , at which quinol oxidation is coupled to proton release and Qi , where quinone reduction is coupled to H+ uptake. Zn2+ is a well established inhibitor of the bovine mitochondrial bc1 complex (1), in which it competes with proton binding (2). In bacterial cyt bc1 complexes Zn2+ has been shown to decelerate electron transfer and transmembrane voltage generation (3). It has been proposed that Zn2+ binds close to the Q0 site, blocking the proton release channel(s). Two Zn2+ binding sites have been located in the avian cyt bc1 complex by X-ray diffraction: one of them, located in a hydrophilic area between the cyt b and c1, might interfere with the egress of H+ (4). To determine the coordination geometry of the bound metal we performed Zn K-edge X-ray Absorption Spectroscopy (XAS) measurements on Zn incubated samples of avian, bovine and bacterial (from Rhodobacter capsulatus (5)) cyt bc1 complexes. Zn stoichiometries were measured by ICP-emission spectroscopy, assuming 5 iron atoms per cyt bc1 complex. Samples were incubated with sub-stoichiometric amounts of Zn to maximize the occupancy of the high affinity site(s) minimizing that of lower affinity site(s). Preliminary analysis of both the avian and the bovine complexes indicates one histidine nitrogen and two oxygen atoms (at 2.00 Å distance) plus one nitrogen or oxygen atom as metal ligands. This agrees with the crystallographic binding site of the avian complex formed by His121, Asp253 and Glu255 (4). First-shell analysis in the bacterial complex suggests two nitrogen and two oxygen atoms (at 2.20 Å) plus one oxygen (or nitrogen) atom as ligands. In order to better define the local structure and possibly locate the cluster of binding residues a multi-shell, multiple scattering analysis is in progress.
14th European Bioenergetics Conference – 2006 Short Reports
169
170
Giachini L.; Francia F.; Lee D-W.; Daldal F.; Huang L-S.; Berry EA.; Cocco T.; Papa S.; Boscherini F.; Venturoli G.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/122286
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