Interactions between heme d and heme b595 in quinol oxidase bd from Escherichia coli: a photoselection study using femtosecond spectroscopy.
Femtosecond spectroscopy was performed on CO-liganded (fully reduced and mixed-valence states) and O(2)-liganded quinol oxidase bd from Escherichia coli. Substantial polarization effects, unprecedented for optical studies of heme proteins, were observed in the CO photodissociation spectra, implying interactions between heme d (the chlorin ligand binding site) and the close-lying heme b(595) on the picosecond time scale; this general result is fully consistent with previous work [Vos, M. H., Borisov, V. B., Liebl, U., Martin, J.-L., and Konstantinov, A. A. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1554-1559]. Analysis of the data obtained under isotropic and anisotropic polarization conditions and additional flash photolysis nanosecond experiments on a mutant of cytochrome bd mostly lacking heme b(595) allow to attribute the features in the well-known but unusual CO dissociation spectrum of cytochrome bd to individual heme d and heme b(595) transitions. This renders it possible to compare the spectra of CO dissociation from reduced and mixed-valence cytochrome bd under static conditions and on a picosecond time scale in much more detail than previously possible. CO binding/dissociation from heme d is shown to perturb ferrous heme b(595), causing induction/loss of an absorption band centered at 435 nm. In addition, the CO photodissociation-induced absorption changes at 50 ps reveal a bathochromic shift of ferrous heme b(595) relative to the static spectrum. No evidence for transient binding of CO to heme b(595) after dissociation from heme d is found in the picosecond time range. The yield of CO photodissociation from heme d on a time scale of < 15 ps is found to be diminished more than 3-fold when heme b(595) is oxidized rather than reduced. In contrast to other known heme proteins, molecular oxygen cannot be photodissociated from the mixed-valence cytochrome bd at all, indicating a unique structural and electronic configuration of the diheme active site in the enzyme.
Borisov
Vitaliy B
VB
A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia.
Liebl
Ursula
U
Rappaport
Fabrice
F
Martin
Jean-Louis
JL
Zhang
Jie
J
Gennis
Robert B
RB
Konstantinov
Alexander A
AA
Vos
Marten H
MH
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D002248:Carbon Monoxide / Q000737:chemistry; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D006418:Heme / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000737:chemistry; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D010100:Oxygen / Q000737:chemistry; D010782:Photolysis; D011485:Protein Binding / Q000235:genetics; D013053:Spectrophotometry / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
D009211:Myoglobin; C018013:carboxymyoglobin; C435883:heme b(595); D018698:Glutamic Acid; D006418:Heme; C080647:heme d; D002248:Carbon Monoxide; D010088:Oxidoreductases; C020094:duroquinol oxidase; D000409:Alanine; D010100:Oxygen