On the oxidation pathways of the mitochondrial bc₁ complex from beef heart

Abstract

We have investigated the oxidation of the reduced ubiquinol : cytochrome c reductase (bcl complex) isolated from beef heart mitochondria. The oxidation of cytochrome cl by both potassium ferricyanide and cytochrome c in the ascorbate-reduced bcl complex is not a first-order process. This is taken as evidence that cytochrome cI is in rapid equilibrium with the Rieske iron-sulphur center. Among the several inhibitors tested, only 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole and stigmatellin are seen to affect this redox equilibrium between the highpotential centers of the beef heart bcl complex.The oxidation of cytochrome b by cytochrome c in both the succinate-reduced and the fully reduced bcl complex is blocked by all the inhibitors tested. This inhibition occurs simultaneously with an acceleration in the oxidation of cytochrome cl, even after extraction of the endogenous ubiquinone which is present in the bcl preparation. Almost complete extraction of ubiquinone from the bcl complex has no effect upon the rapid phase of cytochrome b oxidation, nor does it alter the inhibition of cytochrome b oxidation by the various inhibitors.The oxidation of cytochrome b by exogenous ubiquinones is stimulated by myxothiazol and partially inhibited by antimycin. However, the addition of both these inhibitors together completely blocks the oxidation of cytochrome b by quinones. In contrast, the simultaneous addition of antimycin and myxothiazol has no such synergistic effect upon the oxidation of cytochrome b by cytochrome c.Our data show that intramolecular electron transfer from cytochrome(s) b to the Rieske iron-sulphur center can take place in the bcl complex without involvement of endogenous ubiquinone-10. This electron pathway is sensitive to all the inhibitors of the enzyme. . In addition to these four intrinsic prosthetic groups, a complement of endogenous ubiquinone (mostly ubiquinone-10 in mammals) is usually found in purified preparations of the bcl complex; this quinone is present in an amount which is approximately equimolar with respect to cytochrome c1 Cytochrome c1 and the Rieske iron-sulphur center have their prosthetic groups located in a hydrophilic domain of the complex and are believed to protrude out of the cytoplasmic face of the membrane [I, 21. These redox centers react readily with membrane-impermeant redox mediators such as ferricyanide, ascorbate and the physiological electron acceptor cytochrome c [2]. The latter protein has been shown to react only with cytochrome c1 [2, 71. The 2Fe-2S cluster and c1 are in very rapid redox equilibrium [8,9] and possess a midpoint potential much higher than that of both ubiquinone and the cytochromes b [2,4,9, 101. The use of a variety of specific inhibitors of the bcl complex has been of great value in understanding the observed kinetics of electron transfer [4, 11 -161 (for a review see [12, 171). According to the concepts of the Q-cycle, antimycin, HQNO and funiculosin interact at center ' i ' , whereas a large group of inhibitors including myxothyazol, muci...

Ubiquinol:Cytochrome c Oxidoreductase

“…Moreover, Rich and co-workers (19) have concluded from their recent studies that there is no detectable ubisemiquinone at the Q O site, thus making the proposed chemistry of bifurcated QH 2 oxidation at the Q O site of the Q cycle hypothesis even more complicated. These recent data agree with our previous results (13,14) as well as with those of Palmer and co-workers (20,21) that Q is not an obligatory electron carrier between cytochrome b and ISP/c 1 . In the Q cycle terminology, the complex III inhibitors binding near b H (e.g.…”

Inhibition of the Yeast Cytochrome bc1 Complex by Ilicicolin H, a Novel Inhibitor That Acts at the Qn Site of the bc1 Complex

“…The cytochrome c 1 concentration was determined from the difference spectrum of the ascorbate-reduced versus ferricyanide-oxidized enzyme, using an extinction coefficient of 17.5 mM Ϫ1 cm Ϫ1 at 553-539 nm (12). Cytochrome b concentration was determined from the difference spectrum of the sodium dithionite-reduced minus ascorbate-reduced enzyme using an extinction coefficient of 25 mM Ϫ1 cm Ϫ1 at 563-578 nm (13). The activity of this stock solution of enzyme was stable for a week at 4°C.…”

Mitochondrial cytochrome b: evolution and structure of the protein

“…Correlations between an unusual response to one bc 1 inhibitor and the sequence of the cytochrome b protein are most convincing when they combine sequence analysis in related s p e o e s with the information derived from mutants In the case of the natural resistance of fish to fumculosln [90], the results of a selected screening of fumculosin sensitivity m animal m l t o c h o n d n a suggested that the substitution of the conserved alanme 126 with the bulky m e t h l o n m e in the fish protein (Fig 1) is probably responsible for a substantial increase m the tltre of this inhibitor relatwe to normally sensltwe s p e o e s (Table V and R e f 90) Natural resistance in the plant mItochondnal bc t complex [142] could also be correlated with the exchange of A126 with the bulkier V m the cytochrome b sequence (Fig 2 and R e f 90) The buried location of position 126 within the transmembrane sector of hehx C (Fig 1) may account for the 'hybrid' effects of fumculosln, which effects both center i and center o [90,114,[143][144][145][146] Note that the proteins having a bulky amino a o d at poslt~on 126 also show resistance to U H D B T (Table V), which xs a center o inhibitor that shares with funlculosm the property of effectlng both qulnone sites [113,114,143,144] An interesting property of funlculosln is its remarkable species specificity, even among mammals [143,147] The volume pattern and the comparison of the sequences of sensltwe and resistant species suggested previously that position 194 may also be revolved in funlculosln binding [90] By inspecting the aligned sequences, we noticed that the rabbit protein shows the substitution of alanine 194 with a bulkier vahne residue (Fig 1) Hence, rabbit mltochondrla were expected to be quite resistant to funlculosin, which would explain why rabbits are resistant to this drug in vwo [147] This ~s indeed the case, since the inhibitory potency of funlculosln on the ublqumol cytochrome c reductase actwlty is about 60-fold lower for m l t o c h o n d n a ~solated from rabbit than those from sensltwe mammals (Table V) The cytochrome b proteins of zebra and donkey also have vahne at position 194 (Fig 1 and R e f 32) and differ from that of pig, a...…”