Youssef Hatefi

MEMBRANE TOPOGRAPHY AND NEAR-NEIGHBOR RELATIONSHIPS OF THE MITOCHONDRIAL ATP SYNTHASE SUBUNITS E, F, AND G

The well characterized subunits of the bovine ATP synthase complex are the α, β, γ, δ, and ϵ subunits of the catalytic sector, F1; the ATPase inhibitor protein; and subunits a, b, c, and d, OSCP (oligomycin sensitivity-conferring protein), F6, and A6L, which are present in the membrane sector, F0, and the 45-Å-long stalk that connects F1 to F0. It has been shown recently that bovine ATP synthase preparations also contain three small polypeptides, designated e, f, and g, with respective molecular masses of 8.2, 10.2, and 11.3 kDa. To ascertain their involvement as bona fide subunits of the ATP synthase and to investigate their membrane topography and proximity to the above ATP synthase subunits, polyclonal antipeptide antibodies were raised in the rabbit to the COOH-terminal amino acid residues 57-70 of e, 75-86 of f, and 91-102 of g. It was shown that (i) e, f, and g could be immunoprecipitated with anti-OSCP IgG from a fraction of bovine submitochondrial particles enriched in oligomycin-sensitive ATPase; (ii) the NH2 termini of f and g are exposed on the matrix side of the mitochondrial inner membrane and can be curtailed by proteolysis; (iii) the COOH termini of all three polypeptides are exposed on the cytosolic side of the inner membrane; and (iv) f cross-links to A6L and to g, and e cross-links to g and appears to form an e-e dimer. Thus, the bovine ATP synthase complex appears to have 16 unlike subunits, twice as many as its counterpart in Escherichia coli.

HIGH CYCLIC TRANSHYDROGENASE ACTIVITY CATALYZED BY EXPRESSED AND RECONSTITUTED NUCLEOTIDE-BINDING DOMAINS OF RHODOSPIRILLUM RUBRUM TRANSHYDROGENASE

The hydrophilic, extramembranous domains I (alpha 1 subunit) and III of the Rhodospirillum rubrum nicotinamide nucleotide transhydrogenase were expressed in Escherichia coli and purified therefrom as soluble proteins. These domains bind NAD(H) and NADP(H). respectively, and together they form the enzymes catalytic site. We have demonstrated recently that the isolated domains I and III of the bovine transhydrogenase (or domain I of R. rubrum plus domain III of the bovine enzyme) reconstitute to catalyze transhydrogenation in the absence of the membrane-intercalated domain II, which carries the enzymes proton channel. Here we show that the expressed domains I and III of the R. rubrum transhydrogenase catalyze a very high NADP(H)-dependent cyclic transhydrogenation from NADH to AcPyAD (3-acetylpyridine adenine dinucleotide) with a Vmax of 214 mumol AcPyAD reduced (min x mg of domain I)-1. The reaction mechanism is ping-pong with respect to NADH and AcPyAD, as these nucleotides bind interchangeably to domain I, and the stereospecificity of hydride ion transfer is from the 4A position of NADH to the 4A position of AcPyAD. The expressed domain I is dimeric, like the native alpha 1 subunit of the enzyme, but the expressed domain III is monomeric and contains 0.94 mol NADP(H) per mol.

Energy-transducing nicotinamide nucleotide transhydrogenase: Nucleotide sequences of the genes and predicted amino acid sequences of the subunits of the enzyme fromRhodospirillum rubrum

Based on the amino acid sequence of the N-terminus of the soluble subunit of theRhodospirillum rubrum nicotinamide nucleotide transhydrogenase, two oligonucleotide primers were synthesized and used to amplify the corresponding DNA segment (110 base pairs) by the polymerase chain reaction. Using this PCR product as a probe, one clone with the insert of 6.4kbp was isolated from a genomic library ofR. rubrum and sequenced. This sequence contained three open reading frames, constituting the genesnntA1, nntA2, andnntB of theR. rubrum transhydrogenase operon. The polypeptides encoded by these genes were designated α1, α2, and β, respectively, and are considered to be the subunits of theR. rubrum transhydrogenase. The predicted amino acid sequence of the α1 subunit (384 residues; molecular weight 40276) has considerable sequence similarity to the α subunit of theEscherichia coli and the N-terminal 43-kDa segment of the bovine transhydrogenases. Like the latter, it has a βαβ fold in the corresponding region, and the purified, soluble α 1 subunit cross-reacts with antibody to the bovine N-terminal 43-kDa fragment. The predicted amino acid sequence of the β subunit of theR. rubrum transhydrogenase (464 residues; molecular weight 47808) has extensive sequence identity with the β subunit of theE. coli and the corresponding C-terminal sequence of the bovine transhydrogenases. The chromatophores ofR. rubrum contain a 48-kDa polypeptide, which cross-reacts with antibody to the C-terminal 20-kDa fragment of the bovine transhydrogenase. The predicted amino acid sequence of the α2 subunit of theR. rubrum enzyme (139 residues; molecular weight 14888) has considerable sequence identity in its C-terminal half to the corresponding segments of the bovine and the α subunit of theE. coli transhydrogenases.

Ubiquinol:Cytochrome c Oxidoreductase EFFECTS OF INHIBITORS ON REVERSE ELECTRON TRANSFER FROM THE IRON-SULFUR PROTEIN TO CYTOCHROME b

The effects of inhibitors on the reduction of the bis-heme cytochrome b of ubiquinol: cytochromec oxidoreductase (complex III, bc 1complex) has been studied in bovine heart submitochondrial particles (SMP) when cytochrome b was reduced by NADH and succinate via the ubiquinone (Q) pool or by ascorbate plusN,N,N′,N′-tetramethyl-p-phenylenediamine via cytochrome c 1 and the iron-sulfur protein of complex III (ISP). The inhibitors used were antimycin (an N-side inhibitor), β-methoxyacrylate derivatives, stigmatellin (P-side inhibitors), and ethoxyformic anhydride, which modifies essential histidyl residues in ISP. In agreement with our previous findings, the following results were obtained: (i) When ISP/cytochromec 1 were prereduced or SMP were treated with a P-side inhibitor, the high potential heme b Hwas fully and rapidly reduced by NADH or succinate, whereas the low potential heme b L was only partially reduced. (ii) Reverse electron transfer from ISP/c 1 to cytochrome b was inhibited more by antimycin than by the P-side inhibitors. This reverse electron transfer was unaffected when, instead of normal SMP, Q-extracted SMP containing 200-fold less Q (0.06 mol Q/mol cytochrome b or c 1) were used. (iii) The cytochrome b reduced by reverse electron transfer through the leak of a P-side inhibitor was rapidly oxidized upon subsequent addition of antimycin. This antimycin-induced reoxidation did not happen when Q-extracted SMP were used. The implications of these results on the path of electrons in complex III, on oxidant-induced extra cytochrome b reduction, and on the inhibition of forward electron transfer to cytochrome b by a P-side plus an N-side inhibitor have been discussed.

Ubiquinol:Cytochrome c Oxidoreductase THE REDOX REACTIONS OF THE BIS-HEME CYTOCHROME b IN UNENERGIZED AND ENERGIZED SUBMITOCHONDRIAL PARTICLES

The redox reactions of the bis-heme cytochromeb of the ubiquinol:cytochrome c oxidoreductase complex (complex III, bc 1 complex) were studied in bovine heart submitochondrial particles (SMP). It was shown that (i) when SMP were treated with the complex III inhibitor myxothiazol (or MOA-stilbene or stigmatellin) or with KCN and ascorbate to reduce the high potential centers of complex III (iron-sulfur protein and cytochromes c + c 1), NADH or succinate reduced heme b L slowly and incompletely. In contrast, heme b H was reduced by these substrates completely and much more rapidly. Only when the complex III inhibitor was antimycin, and the high potential centers were in the oxidized state, NADH or succinate was able to reduce bothb H and b L rapidly and completely. (ii) When NADH or succinate was added to SMP inhibited at complex III by antimycin and energized by ATP, the bis-heme cytochromeb was reduced only partially. Prereduction of the high potential centers was not necessary for this partial breduction, but slowed down the reduction rate. Deenergization of SMP by uncoupling (or addition of oligomycin to inhibit ATP hydrolysis) resulted in further b reduction. Addition of ATP afterb was reduced by substrate resulted in partialb oxidation, and the heme remaining reduced appeared to be mainly b L. Other experiments suggested that the redox changes of cytochrome b effected by energization and deenergization of SMP occurred via electronic communication with the ubiquinone pool. These results have been discussed in relation to current concepts regarding the mechanism of electron transfer by complex III.

Chapter 11 The energy-transducing nicotinamide nucleotide transhydrogenase

Publisher Summary This chapter discusses the energy-transducing nicotinamide nucleotide transhydrogenase. The energy-transducing nicotinamide nucleotide transhydrogenases of mitochondria and bacteria are membrane-bound enzymes that catalyze the direct and stereospecific transfer of a hydride ion between the 4A position of NAD (H) and the 4B position of NADP (H). In the mitochondria, the enzyme is embedded in the inner membrane, with its nucleotide binding sites protruding into the matrix, and the transhydrogenation reaction is coupled to transmembrane proton translocation with a H + / H - stoichiometry of unity. Because of the stereospecificity of hydride ion transfer, this type of transhydrogenase is referred to as “AB-transhydrogenase.” The known AB-transhydrogenases are integral membrane proteins, their scalar transhydrogenation reaction is coupled to proton translocation, and they contain separate binding sites for NAD (H) and NADP (H), and have no prosthetic groups. The BB-type transhydrogenases are water-soluble, do not pump protons, are flavoproteins containing FAD, have a single substrate-binding site, and are thought to be concerned with equilibrating the cellular NAD (H) and NADP (H) pools.

The Mitochondrial Enzymes of Oxidative Phosphorylation

Today, the amino acid sequences of proteins of unknown function are deduced from the DNA before they are isolated, the receptors of unknown hormones are identified, and antibodies that catalyze nonbiological reactions are generated. Forty years ago, when this author began work on the mitochondrial oxidative phosphorylation system, it was necessary to have a well-defined and assayable biological reaction before one could attempt to isolate the enzyme(s) that catalyzed it. The fortunate circumstance that led to the discovery of the respiratory chain enzyme complexes occurred in 1957, when F. L. Crane, R. L. Lester, C. Widmer, and the author isolated coenzyme Q (ubiquinone) from bovine heart mitochondria and obtained evidence for its role as a respiratory chain electron carrier (Hatefi, 1963; Crane et al., 1957). Consequently, one could think of the mitochondrial electron transport system in terms of the following four assayable reactions, and one could attempt to isolate the four enzymes or enzyme systems that catalyzed them: n n

Studies on the Structure of the Mitochondrial ATP Synthase Complex and the Mechanism of ATP Synthesis

{text{NADH + Q + }}{{{text{H}}}^{ + }}{text{ }} rightleftharpoons {text{ NA}}{{{text{D}}}^{ + }}{text{ + Q}}{{{text{H}}}_{2}}