TY - JOUR
T1 - Heme-heme and heme-ligand interactions in the di-heme oxygen-reducing site of cytochrome bd from Escherichia coli revealed by nanosecond absorption spectroscopy
AU - Rappaport, Fabrice
AU - Zhang, Jie
AU - Vos, Marten H.
AU - Gennis, Robert B.
AU - Borisov, Vitaliy B.
N1 - This work was supported by the Russian Foundation for Basic Research (to V.B.B., grant 08-04-00093 ). V.B.B. was recipient of a FEBS short-term fellowship. We thank Professor Pierre Joliot (Paris) and Dr. Alexander Konstantinov (Moscow) for their interest and stimulating discussions of this work.
PY - 2010/9
Y1 - 2010/9
N2 - Cytochrome bd is a terminal quinol:O2 oxidoreductase of respiratory chains of many bacteria. It contains three hemes, b558, b595, and d. The role of heme b595 remains obscure. A CO photolysis/recombination study of the membranes of Escherichia coli containing either wild type cytochrome bd or inactive E445A mutant was performed using nanosecond absorption spectroscopy. We compared photoinduced changes of heme d-CO complex in one-electron-reduced, two-electron-reduced, and fully reduced states of cytochromes bd. The line shape of spectra of photodissociation of one-electron-reduced and two-electron-reduced enzymes is strikingly different from that of the fully reduced enzyme. The difference demonstrates that in the fully reduced enzyme photolysis of CO from heme d perturbs ferrous heme b595 causing loss of an absorption band centered at 435nm, thus supporting interactions between heme b595 and heme d in the di-heme oxygen-reducing site, in agreement with previous works. Photolyzed CO recombines with the fully reduced enzyme monoexponentially with Τ~12μs, whereas recombination of CO with one-electron-reduced cytochrome bd shows three kinetic phases, with Τ~14ns, 14μs, and 280μs. The spectra of the absorption changes associated with these components are different in line shape. The 14ns phase, absent in the fully reduced enzyme, reflects geminate recombination of CO with part of heme d. The 14-μs component reflects bimolecular recombination of CO with heme d and electron backflow from heme d to hemes b in ~4% of the enzyme population. The final, 280-μs component, reflects return of the electron from hemes b to heme d and bimolecular recombination of CO in that population. The fact that even in the two-electron-reduced enzyme, a nanosecond geminate recombination is observed, suggests that namely the redox state of heme b595, and not that of heme b558, controls the pathway(s) by which CO migrates between heme d and the medium.
AB - Cytochrome bd is a terminal quinol:O2 oxidoreductase of respiratory chains of many bacteria. It contains three hemes, b558, b595, and d. The role of heme b595 remains obscure. A CO photolysis/recombination study of the membranes of Escherichia coli containing either wild type cytochrome bd or inactive E445A mutant was performed using nanosecond absorption spectroscopy. We compared photoinduced changes of heme d-CO complex in one-electron-reduced, two-electron-reduced, and fully reduced states of cytochromes bd. The line shape of spectra of photodissociation of one-electron-reduced and two-electron-reduced enzymes is strikingly different from that of the fully reduced enzyme. The difference demonstrates that in the fully reduced enzyme photolysis of CO from heme d perturbs ferrous heme b595 causing loss of an absorption band centered at 435nm, thus supporting interactions between heme b595 and heme d in the di-heme oxygen-reducing site, in agreement with previous works. Photolyzed CO recombines with the fully reduced enzyme monoexponentially with Τ~12μs, whereas recombination of CO with one-electron-reduced cytochrome bd shows three kinetic phases, with Τ~14ns, 14μs, and 280μs. The spectra of the absorption changes associated with these components are different in line shape. The 14ns phase, absent in the fully reduced enzyme, reflects geminate recombination of CO with part of heme d. The 14-μs component reflects bimolecular recombination of CO with heme d and electron backflow from heme d to hemes b in ~4% of the enzyme population. The final, 280-μs component, reflects return of the electron from hemes b to heme d and bimolecular recombination of CO in that population. The fact that even in the two-electron-reduced enzyme, a nanosecond geminate recombination is observed, suggests that namely the redox state of heme b595, and not that of heme b558, controls the pathway(s) by which CO migrates between heme d and the medium.
KW - Chlorin
KW - Cytochrome
KW - Gas molecule
KW - Ligand binding
KW - Photobiology
KW - Respiration
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U2 - 10.1016/j.bbabio.2010.05.010
DO - 10.1016/j.bbabio.2010.05.010
M3 - Article
C2 - 20529691
AN - SCOPUS:77954761715
SN - 0005-2728
VL - 1797
SP - 1657
EP - 1664
JO - Biochimica et Biophysica Acta - Bioenergetics
JF - Biochimica et Biophysica Acta - Bioenergetics
IS - 9
ER -