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V B Borisov

Publications and source records attributed to V B Borisov.

7 recordsLinked to original sources
PubMed2001

Interaction of cytochrome bd with carbon monoxide at low and room temperatures: evidence that only a small fraction of heme b595 reacts with CO.

Azotobacter vinelandii is an obligately aerobic bacterium in which aerotolerant dinitrogen fixation requires cytochrome bd. This oxidase comprises two polypeptide subunits and three hemes, but no copper, and has been studied extensively. However, there remain apparently conflicting reports on the reactivity of the high spin heme b(595) with ligands. Using purified cytochrome bd, we show that absorption changes induced by CO photodissociation from the fully reduced cytochrome bd at low temperatures demonstrate binding of the ligand with heme b(595). However, the magnitude of these changes corresponds to the reaction with CO of only about 5% of the heme. CO binding with a minor fraction of heme b(595) is also revealed at room temperature by time-resolved studies of CO recombination. The data resolve the apparent discrepancies between conclusions drawn from room and low temperature spectroscopic studies of the CO reaction with cytochrome bd. The results are consistent with the proposal that hemes b(595) and d form a diheme oxygen-reducing center with a binding capacity for a single exogenous ligand molecule that partitions between the hemes d and b(595) in accordance with their intrinsic affinities for the ligand. In this model, the affinity of heme b(595) for CO is about 20-fold lower than that of heme d.

Carbon Monoxide↗
PubMed2000

Electrogenic reactions of cytochrome bd.

Cytochrome bd is one of the two terminal quinol oxidases in the respiratory chain of Escherichia coli. The enzyme catalyzes charge separation across the bacterial membrane during the oxidation of quinols by dioxygen but does not pump protons. In this work, the reaction of cytochrome bd with O(2) and related reactions has been studied by time-resolved spectrophotometric and electrometric methods. Oxidation of the fully reduced enzyme by oxygen is accompanied by rapid generation of membrane potential (delta psi, negative inside the vesicles) that can be described by a two-step sequence of (i) an initial oxygen concentration-dependent, electrically silent, process (lag phase) corresponding to the formation of a ferrous oxy compound of heme d and (ii) a subsequent monoexponential electrogenic phase with a time constant <60 mus that matches the formation of ferryl-oxo heme d, the product of the reaction of O(2) with the 3-electron reduced enzyme. No evidence for generation of an intermediate analogous to the "peroxy" species of heme-copper oxidases could be obtained in either electrometric or spectrophotometric measurements of cytochrome bd oxidation or in a spectrophotometric study of the reaction of H(2)O(2) with the oxidized enzyme. Backflow of electrons upon flash photolysis of the singly reduced CO complex of cytochrome bd leads to transient generation of a delta psi of the opposite polarity (positive inside the vesicles) concurrent with electron flow from heme d to heme b(558) and backward. The amplitude of the delta psi produced by the backflow process, when normalized to the reaction yield, is close to that observed in the direct reaction during the reaction of fully reduced cytochrome bd with O(2) and is apparently associated with full transmembrane translocation of approximately one charge.

Carbon Monoxide↗
PubMed2000

Femtosecond resolution of ligand-heme interactions in the high-affinity quinol oxidase bd: A di-heme active site?

Interaction of the two high-spin hemes in the oxygen reduction site of the bd-type quinol oxidase from Escherichia coli has been studied by femtosecond multicolor transient absorption spectroscopy. The previously unidentified Soret band of ferrous heme b(595) was determined to be centered around 440 nm by selective excitation of the fully reduced unliganded or CO-bound cytochrome bd in the alpha-band of heme b(595). The redox state of the b-type hemes strongly affects both the line shape and the kinetics of the absorption changes induced by photodissociation of CO from heme d. In the reduced enzyme, CO photodissociation from heme d perturbs the spectrum of ferrous cytochrome b(595) within a few ps, pointing to a direct interaction between hemes b(595) and d. Whereas in the reduced enzyme no heme d-CO geminate recombination is observed, in the mixed-valence CO-liganded complex with heme b(595) initially oxidized, a significant part of photodissociated CO does not leave the protein and recombines with heme d within a few hundred ps. This caging effect may indicate that ferrous heme b(595) provides a transient binding site for carbon monoxide within one of the routes by which the dissociated ligand leaves the protein. Taken together, the data indicate physical proximity of the hemes d and b(595) and corroborate the possibility of a functional cooperation between the two hemes in the dioxygen-reducing center of cytochrome bd.

Binding Sites↗
PubMed1996

[Cytochrome bd: structure and properties].

Literary evidence concerning the arrangement and functioning of the cytochrome bd complex is reviewed with particular emphasis on ligand-binding properties of the enzyme. Some novel data on cytochrome bd interaction with carbon monoxide, cyanide and hydrogen peroxide are presented.

Cytochrome b Group↗
PubMed1995

[Interaction of Escherichia coli cytochrome bd with hydrogen peroxide].

The absorption spectrum of the cytochrome bd complex from Escherichia coli in the "as isolated" state is characterized by an intense band at approximately 648 nm belonging to reduced heme d oxycomplex (d2+-O2). This band is often accompanied by a small shoulder around 680 nm. Treatment of the oxycomplex with hydrogen peroxide results in the loss of the 648 nm band and increased absorbance at 680 nm. The peak at 680 nm also appears in the difference absorption spectrum after addition of hydrogen peroxide to the oxidized form of the enzyme and can be attributed to formation of a peroxy or an oxoferryl complex of heme d. The increase in extinction at 680 nm is accompanied by a small red shift of the Soret band; the corresponding difference spectrum with lambda min = 405-410 nm and lambda max = 430-440 nm is of a magnitude similar to the changes in the visible region (delta A440-410 approximately equals 10 mM-1.cm-1). This circumstance favours H2O2 interaction with heme d rather than b595. The lineshape of the H2O2-induced spectral changes does not vary throughout the hydrogen peroxide concentration range studied (5 microM-5 mM). The H2O2 concentration dependence on the 680 nm peak magnitude follows a saturation curve with apparent Kd of 30-40 microM. The product of cytochrome bd interaction with H2O2 reacts with cyanide approximately tenfold slower than the free oxidized enzyme. Addition of excess catalase to the hydrogen peroxide-treated cytochrome bd complex fully reverses the H2O2-induced spectral changes. However, the rate of disappearance of these changes (keff approximately equals 10(-3) s-1) is ca. 10-fold slower than expected for the dissociation rate constant, koff, for the peroxy adduct, assuming reversible H2O2 binding with Kd approximately equal to 30-40 microM and kon > 500 M-1.s-1. This may point to H2O2 interaction with cytochrome bd, being essentially irreversible. The initial addition of H2O2 to heme d is likely to be followed by cleavage of the O--O bond, giving rise to the oxoferryl state (Fe4+ = O) of heme d which disappears upon removal of H2O2 by catalase due to reduction by endogenous electron sources.

Cytochrome b Group↗
PubMed1994

[Oxygenated cytochrome bd from Escherichia coli could be transformed into an oxidized form by lipophilic electron acceptors].

The cytochrome bd complex as isolated from Escherichia coli under aerobic conditions is in a stable oxygenated form [formula: see text], characterized by an intense peak at 650 nm in an absolute absorption spectrum. The commonly used oxidants ferricyanide and persulfate have no effect on the oxygenated form, whereas the addition of lipophilic electron acceptors, such as tetrachlorobenzoquinone or ferricinium, results in the decay of the heme d oxy-complex and the enzyme transition into the fully oxidized form, [formula: see text]. Interaction of the oxygenated cytochrome bd complex with both tetrachlorobenzoquinone and ferricinium is suppressed by pentachlorophenol, an inhibitor of the enzyme ubiquinol oxidase activity. It is suggested that redox centers of cytochrome bd reside in the hydrophobic environment which can prevent their interaction with the hydrophilic oxidants.

Chloranil↗