scholarly journals The iron(III)-adriamycin complex inhibits cytochrome c oxidase before its inactivation

1988 ◽  
Vol 250 (3) ◽  
pp. 827-834 ◽  
Author(s):  
B B Hasinoff ◽  
J P Davey
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Competitive Inhibition ◽  
Enzyme Inhibitor ◽  
Kinetic Studies ◽  
Slow Inactivation ◽  
Radical Species ◽  
Inhibitor Complex ◽  
Antitumour Drug ◽  
Strong Contrast

Cytochrome c oxidase was found to be competitively inhibited by a complex formed between Fe3+ and the cardiotoxic antitumour drug adriamycin (doxorubicin) with an inhibition constant, Ki, of 12 microM. This competitive inhibition precedes the slower Fe3+-adriamycin induced inactivation of cytochrome c oxidase. In strong contrast with this result, free adriamycin was not observed to either inhibit or inactivate cytochrome c oxidase (Ki greater than 3 mM). Since, typically, polycations are known to inhibit cytochrome c oxidase, the competitive inhibition displayed by the Fe3+-adriamycin complex may also result from its polycationic character. Cytochrome c oxidase was also inhibited by pentan-1-ol (Ki 13 mM), and kinetic studies carried out in the presence of both inhibitors demonstrated that the Fe3+-adriamycin complex and pentan-1-ol are mutually exclusive inhibitors of cytochrome c oxidase. The inhibitor pentan-1-ol was also effective in preventing the slow inactivation of cytochrome c oxidase induced by Fe3+-adriamycin, presumably by blocking its binding to the enzyme. It is postulated that the slow inactivation of cytochrome c oxidase occurs when reactive radical species are produced while the Fe3+-adriamycin is complexed to cytochrome c oxidase in an enzyme-inhibitor complex. The Fe3+-adriamycin-induced inactivation of cytochrome c oxidase may be, in part, responsible for the cardiotoxicity of adriamycin.

scholarly journals Mechanism in the reaction of cytochrome c oxidase with organic hydroperoxides: an ESR spin-trapping investigation

2002 ◽  
Vol 365 (2) ◽  
pp. 461-469 ◽  
Author(s):  
Yeong-Renn CHEN ◽  
Ronald P. MASON
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Spin Trap ◽  
Spin Trapping ◽  
Initial Reaction ◽  
Radical Species ◽  
Alkoxyl Radical ◽  
Tyrosine Residues ◽  
Esr Spin Trapping ◽  
Organic Hydroperoxides

Organic hydroperoxides are of great utility in probing the reaction mechanism and the toxicological consequences of lipid peroxidation. In the present study, ESR spin-trapping was employed to investigate the peroxidation of mitochondrial cytochrome c oxidase (CcO) with t-butyl hydroperoxide (t-BuOOH) and cumene hydroperoxide (CumOOH). The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to detect the radical species formed from the reaction of CcO with t-BuOOH. The presence of t-BuOOH-derived alkoxyl radical (t-BuO˙) as the primary radical indicates reductive scission of the O—O bond by CcO. The ESR signal of DMPO/˙Ot-Bu can be partially abolished by cyanide, implying that the reductive cleavage involved the haem a3CuB binuclear site of CcO. A nitroso spin trap, 2-methyl-2-nitrosopropane (MNP), was used to detect and identify radical species from the reaction of CcO with CumOOH. In addition to the t-BuOOH-derived methyl, hydroxylmethyl and tertiary carbon-centred radicals, a protein-derived radical was detected. The intensity of the ESR signal from the protein radical increased with the CumOOH concentration at low CumOOH/CcO ratios, with maximal intensity at a ratio of 100mol of CumOOH/mol of CcO. The immobilized protein radical adduct of MNP was stable and persistent after dialysis; it was also resistant to proteolytic digestion, suggesting that it was formed in the transmembrane region, a region that is not accessible to proteases. Its signal was greatly enhanced when CcO cysteine residues were chemically modified by N-ethylmaleimide, when the tryptophan residues in CcO were oxidized by N-bromosuccimide, and when tyrosine residues on the surface of CcO were iodinated, showing that a radical equilibrium was established among the cysteine, tryptophan and tyrosine residues of the protein-centred radical. Pre-treatment of CcO with cyanide prevented detectable MNP adduct formation, confirming that the haem a3-CuB binuclear centre was the initial reaction site. When the CcO was pre-treated with 10mM (100 equivalents) of CumOOH, the enzyme activity decreased by more than 20%. This inhibition was persistent after dialysis, suggesting that the detected protein-centred radical was, in part, involved in the irreversible inactivation by CumOOH. Visible spectroscopic analysis revealed that the haem a of CcO was not affected during the reaction. However, the addition of pyridine to the reaction mixture under alkaline conditions resulted in the destruction of the haem centre of CcO, suggesting that its protein matrix rather than its haem a is the target of oxidative damage by the organic hydroperoxide.

Kinetic Studies on Mammalian Cytochrome c Oxidase

1973 ◽  
Vol 1 (1) ◽  
pp. 34-35 ◽  
Author(s):  
E. ANTONINI ◽  
M. BRUNORI ◽  
C. GREENWOOD ◽  
M. T. WILSON
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Kinetic Studies

scholarly journals Transient kinetic studies of Neurospora crassa cytochrome c oxidase

1983 ◽  
Vol 215 (2) ◽  
pp. 425-427 ◽  
Author(s):  
M Brunori ◽  
M C Silvestrini ◽  
M T Wilson ◽  
H Weiss
Keyword(s):  
Cytochrome C ◽  
Neurospora Crassa ◽  
Cytochrome C Oxidase ◽  
Flash Photolysis ◽  
Kinetic Studies ◽  
Dissociation Rate ◽  
Steady State Kinetic ◽  
Kinetic Investigations ◽  
Dissociation Rate Constants

The reaction of Neurospora crassa cytochrome c oxidase with CO was studied by flash-photolysis and rapid-mixing experiments, leading to the determination of the association and dissociation rate constants (7 X 10(4) M-1 X s-1 and 0.02s-1 respectively). Pre-steady-state kinetic investigations of the catalytic properties of the enzyme showed that under proper conditions Neurospora cytochrome c oxidase can be ‘pulsed’, i.e. activated, like the mammalian enzyme. The ‘pulsed’ species is spectroscopically different from the ‘resting’ one, and the decay into the ‘resting’ state is fast (t1/2 approx. 3 min).

scholarly journals Kinetic studies on the reaction between cytochrome c oxidase and ferrocytochrome c

1975 ◽  
Vol 147 (1) ◽  
pp. 145-153 ◽  
Author(s):  
M T Wilson ◽  
C Greenwood ◽  
M Brunori ◽  
E Antonini
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Time Course ◽  
Kinetic Studies ◽  
Initial Reaction ◽  
Fast Phase ◽  
Ferrocytochrome C ◽  
Cytochrome C2 ◽  
Kinetics Of ◽  
Flow Experiments

In stopped-flow experiments in which oxidized cytochrome c oxidase was mixed with ferrocytochrome c in the presence of a range of oxygen concentrations and in the absence and presence of cyanide, a fast phase, reflecting a rapid approach to an equilibrium, was observed. Within this phase, one or two molecules of ferrocytochrome were oxidized per haem group of cytochrome a, depending on the concentration of ferrocytochrome c used. The reasons for this are discussed in terms of a mechanism in which all electrons enter through cytochrome a, which, in turn, is in rapid equilibrium with a second site, identified with ‘visible’ copper (830 nm-absorbing) Cud (Beinert et al., 1971). The value of the bimolecular rate constant for the reaction between cytochromes c2+ and a3+ was between 10(6) and 10(7) M(-1)-S(-1); some variability from preparation to preparation was observed. At high ferrocytochrome c concentrations, the initial reaction of cytochrome c2+ with cytochrome a3+ could be isolated from the reaction involving the ‘visible’ copper and the stoicheiometry was found to approach one molecule of cytochrome c2+ oxidized for each molecule of cytochrome a3+ reduced. At low ferrocytochrome c concentrations, however, both sites (i.e. cytochrome a and Cud) were reduced simultaneously and the stoicheiometry of the initial reaction was closer to two molecules of cytochrome c2+ oxidized per molecule of cytochrome a reduced. The bleaching of the 830 nm band lagged behind or was simultaneous with the formation of the 605 nm band and does not depend on the cytochrome c concentration, whereas the extinction at the steady-state does. The time-course of the return of the 830 nm-absorbing species is much faster than the bleaching of the 605 nm-absorbing component, and parallels that of the turnover phase of cytochrome c2+ oxidation. Additions of cyanide to the oxidase preparations had no effect on the observed stoicheiometry or kinetics of the reduction of cytochrome a and ‘visible’ copper, but inhibited electron transfer to the other two sites, cytochrome a3 and the undetectable copper, Cuu.

scholarly journals Inactivation of Δ5-3-oxo steroid isomerase with active-site-directed acetylenic steroids

1981 ◽  
Vol 193 (1) ◽  
pp. 217-227 ◽  
Author(s):  
T M Penning ◽  
D F Covey ◽  
P Talalay
Keyword(s):  
Active Site ◽  
Competitive Inhibition ◽  
Enzyme Inhibitor ◽  
Covalent Bond ◽  
Addition Product ◽  
Ring Structure ◽  
Dependent Manner ◽  
Compound I ◽  
Inhibitor Complex ◽  
Acetylenic Ketone

Several steroid analogues containing conjugated acetylenic ketone groups as part of a seco-ring structure or as substituents on the intact steroid system are irreversible inhibitors of delta 5-3-oxo steroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni. Thus 10 beta-(1-oxoprop-2-ynyl)oestr-4-ene-3,17-dione (I), 5,10-seco-oestr-4-yne-3,10,17-trione (II), 17 beta-hydroxy-5,10-seco-oestr-4-yne-3,10-dione (III) and 17 beta-(1-oxoprop-2-ynyl)androst-4-en-3-one (IV) irreversibly inactivate isomerase in a time-dependent manner. In all cases saturation kinetics are observed. Protection against inactivation is afforded by the powerful competitive inhibitor 19-nortestosterone. The inhibition constants (Ki) for 19-nortestosterone obtained from such experiments are in good agreement with those determined from conventional competitive-inhibition studies of enzyme activity. These compounds thus appear to be active-site directed. In every case the inactivated enzyme could be dialysed without return of activity, indicating that a stable covalent bond probably had formed between the steroid and enzyme. Compound (I) is a very potent inhibitor of isomerase [Ki = 66.0 microM and k+2 = 12.5 × 10(-3) s-1 (where Ki is the dissociation constant of the reversible enzyme-inhibitor complex and k+2 is the rate constant for the inactivation reaction of the enzyme-inhibitor complex)] giving half-lives of inactivation of 30-45 s at saturation. It is argued that the basic-amino-acid residue that abstracts the intramolecularly transferred 4 beta-proton in the reaction mechanism could form a Michael-addition product with compound (I). In contrast, although compound (IV) has a lower inhibition constant (Ki = 14.5 microM), it is a relatively poor alkylating agent (k+2 = 0.13 × 10(-3) s-1). If the conjugated acetylenic ketone groups are replaced by alpha-hydroxyacetylene groups, the resultant analogues of steroids (I)-(IV) are reversible competitive inhibitors with Ki values in the range 27-350 microM. The enzyme binds steroids in the C19 series with functionalized acetylenic substituents at C-17 in preference to steroids in the C18 series bearing similar groups in the ring structure or as C-10 substituents. In the 5,10-seco-steroid series the presence of hydroxy groups at both C-3 and C-17 is deleterious to binding by the enzyme.

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