News and Publications

Bioenergetics of Mitochondria and Bacteria

Y. Hatefi, A. Matsuno-Yagi, M. Yamaguchi

In most aerobic organisms, the terminal oxidation of foodstuffs, the capture of the energy derived from this oxidation, and the use of the energy for ATP synthesis are accomplished by 5 enzyme complexes, which are associated with the inner membrane of mitochondria in eukaryotes and the plasma membrane in prokaryotes. The 5 complexes are NADH:quinone oxidoreductase (complex I), succinate:quinone oxidoreductase (complex II), quinol:cytochrome c (c₂, plastocyanin) oxidoreductase (complex III), cytochrome c oxidase (complex IV), and ATP synthase (complex V). Our research focuses on the composition, structure, and interaction of these enzyme complexes; the mechanisms of electron transfer by complexes I, II, and III; proton translocation by complexes I, III, and V; and ATP synthesis and hydrolysis by complex V.

UBIQUINOL:CYTOCHROME C OXIDOREDUCTASE

Earlier studies indicated that several electron-transfer features of ubiquinol:cytochrome c oxidoreductase in bovine submitochondrial particles were incompatible with the generally accepted ubiquinone cycle (Q cycle) hypothesis of electron transfer and proton translocation mediated by this enzyme (Fig. 1B). Briefly, we showed the following: (1) In submitochondrial particles, reoxidation of the bis-heme cytochrome b of complex III could be inhibited by either antimycin or myxothiazol. The inhibition by either reagent was incomplete, and oxidation of heme b_L (b₅₆₆) through the leak allowed by either inhibitor was at least 10 times faster than oxidation of heme b_H (b₅₆₂). (2) Cytochrome b of complex III could be partially reduced via the Rieske iron-sulfur protein and cytochrome c₁ (ISP/c₁) by ascorbate or, faster and to a greater extent, by ascorbate plus N,N,N´,N´-tetramethyl-p-phenylenediamine (TMPD). This reaction was inhibited more strongly by antimycin than by myxothiazol. (3) Ascorbate or ascorbate plus TMPD could also partially reduce cytochrome b in ubiquinone-depleted bovine heart submitochondrial particles and in submitochondrial particles from a ubiquinone-deficient yeast mutant. (4) Antimycin and myxothiazol, which exert their maximal inhibition at concentrations stoichiometric to complex III monomer, each inhibited 3 reactions of the bis-heme cytochrome b, all incompletely. The strongest effect of either reagent was inhibition of electron transfer from heme b_H to heme b_L; next was inhibition of the reoxidation of heme b_L via ISP/c₁; least was inhibition of substrate (NADH or succinate) reduction of cytochrome b, which is known to require the combined actions of both antimycin and myxothiazol. In these regards, 2-n-heptyl-4-hydroxyquinoline-N-oxide behaved similarly to antimycin, and (E)-methyl-3-methoxy-2-(4-trans-stibenyl)acrylate and stigmatellin behaved similarly to myxothiazol. Oxidation of reduced hemes b_H and b_L or their reduction by reverse electron transfer via ISP/c₁ was not inhibited to a greater extent when submitochondrial particles were treated with both antimycin and myxothiazol. These results are summarized in Figure 1A.

However, an important feature of complex III electron transfer, the oxidant-induced extrareduction of cytochrome b, had not yet been investigated. This feature was a major consideration for the design of the Q cycle in 1975. Studies by Rieske in 1971 indicated that prereduction of ISP/c₁ of complex III prevented the reduction of cytochrome b by reduced ubiquinone in antimycin-treated complex III. However, when ISP/c₁ was reoxidized by the addition of potassium ferricyanide, cytochrome b became rapidly reduced. The Q cycle hypothesis easily accounts for this effect, because according to this hypothesis ubiquinol must first be oxidized by 1 electron via ISP/c₁ to form ubisemiquinone, which is the reductant of cytochrome b. Prereduction of ISP/c₁ prevents the 1-electron oxidation of ubiquinol to yield the electron donor to cytochrome b. Our finding that ubiquinone was not required for electron transfer between cytochromes b and c₁ indicated, however, that oxidant-induced extrareduction of cytochrome b required a different explanation, and our experiment strongly supported this conclusion.

We found that in antimycin-treated submitochondrial particles, prereduction of ISP/c₁ with ascorbate with or without TMPD did not prevent the subsequent reduction of cytochrome b with NADH or succinate. Heme b_H was fully and rapidly reduced, but the reduction of heme b_L was slow and incomplete. Addition of ferricyanide at the beginning of the slow phase resulted in rapid reduction of heme b_L. When the inhibitor was myxothiazol instead of antimycin, a similar biphasic reduction of cytochrome b by NADH or succinate occurred, regardless of whether ISP/c₁ was prereduced or not. Other experiments showed that prereduction of ISP/c₁ or the presence of myxothiazol affected only the reduction of heme b_L and that heme b_H remained in rapid electronic communication with complexes I and II. Therefore, the phenomenon of oxidant-induced extrareduction of cytochrome b means that the reduced state of ISP/c₁ interfered somehow with substrate reduction of heme b_L only. Recent studies of others have indicated that in the presence of myxothiazol, the iron-sulfur protein moves close to heme b_L. Perhaps reduced iron-sulfur protein also sits close to heme b_L, and this proximity interferes with the rapid and complete reduction of this heme by substrates.

We also examined the effect of membrane energization on the kinetics of cytochrome b reduction and oxidation. When NADH or succinate was added to submitochondrial particles inhibited at complex III by antimycin and energized by ATP, the bis-heme cytochrome b was still reduced, albeit partially. Prereduction of the high-potential centers was not necessary for this partial reduction of cytochrome b but did slow the reduction rate. Deenergization of submitochondrial particles by uncoupling resulted in further reduction of cytochrome b. Addition of ATP after cytochrome b was reduced by substrate resulted in partial oxidation of the cytochrome, and the heme remaining reduced appeared to be mainly heme b_L.

The important point is that again prereduction of ISP/c₁ in antimycin- and ATP-treated submitochondrial particles did not inhibit the subsequent reduction of cytochrome b by NADH or succinate. The only difference from the results obtained with unenergized submitochondrial particles was that in energized submitochondrial particles, heme b_L appeared to be reduced faster than was heme b_H, a finding that may be referable to the reversal of membrane polarity under energized conditions and to an increase in the E_m of heme b_L in these particles.

MITOCHONDRIAL NADH:UBIQUINONE OXIDOREDUCTASE

We showed that treatment of bovine mitochondrial complex I (NADH:ubiquinone oxidoreductase) with NADH or NADPH, but not with NAD or NADP, increases the susceptibility of a number of subunits to tryptic degradation. This increased susceptibility involved subunits that contain electron carriers, such as FMN and iron-sulfur clusters, and subunits that lack electron carriers. Thus, reduction of complex I by NADH or NADPH increased the trypsin susceptibility of the 51- and the 24-kD subunits of the primary dehydrogenase of the flavoprotein domain; the 75-, 49-, and the 30-kD subunits of the iron-protein domain; and the 23-kD subunit of the hydrophobic protein domain.

Alterations in the cross-linking pattern of complex I subunits that occurred when the enzyme was pretreated with NADH or NADPH also indicated that complex I undergoes extensive changes in conformation when reduced by substrate. Furthermore, we previously showed that in submitochondrial particles the affinity of complex I for NAD increased by 20-fold or more in electron transfer from succinate to NAD when the particles were energized by ATP hydrolysis. Together, these results suggest that energy coupling in complex I may involve changes in protein conformation as a key step.

We also showed that treatment of complex I with trypsin in the presence of NADPH, but not NADH or NAD(P), produced from the 39-kD subunit a 33-kD degradation product that resisted further hydrolysis. Like the 39-kD subunit, the 33-kD product bound to a NADP-agarose affinity column and could be eluted with a buffer containing NADPH. Perhaps, together with the acyl carrier protein of complex I, the NADP(H)-binding 39-kD subunit is involved in intramitochondrial synthesis of fatty acids.

PUBLICATIONS

Hatefi, Y. The mitochondrial enzymes of oxidative phosphorylation. In: Frontiers of Cellular Bioenergetics: Molecular Biology, Biochemistry and Physiopathology. Papa, S., Guerrieri, F., Tager, J. (Eds.). Plenum, New York, in press.

Hatefi, Y. Reconstitution of the electron transport system of bovine heart mitochondria. In: Biomembranes. Packer, L., Fleischer, S. (Eds.). Academic Press, San Diego, 1997, p. 605.

Matsuno-Yagi, A., Hatefi, Y. Ubiquinol:cytochrome c oxidoreductase: The redox reactions of the bis-heme cytochrome b in unenergized and energized submitochondrial particles. J. Biol. Chem. 272:16928, 1997.

Yamaguchi, M., Belogrudov, G.I., Hatefi, Y. Mitochondrial NADH-ubiquinone oxidoreductase (complex I): Effect of substrates on the fragmentation of subunits by trypsin. J. Biol. Chem. 273:8094, 1998.