The central active site arginine in sulfite oxidizing enzymes alters kinetic properties by controlling electron transfer and redox interactions

A central conserved arginine, first identified as a clinical mutation leading to sulfite oxidase deficiency, is essential for catalytic competency of sulfite oxidizing molybdoenzymes, but the molecular basis for its effects on turnover and substrate affinity have not been fully elucidated.We have used a bacterial sulfite dehydrogenase, SorT, which lacks an internal heme group, but transfers electrons to an external, electron accepting cytochrome, SorU, to investigate the molecular functions of this arginine residue (Arg78). Assay of the SorT Mo centre catalytic competency in the absence of SorU showed that substitutions in the central arginine (R78Q, R78K and R78M mutations) only moderately altered SorT catalytic properties, except for R78M which caused significant reduction in SorT activity. The substitutions also altered the Mo-centre redox potentials (Mo^VI/V potential lowered by ca. 60–80 mV). However, all Arg78 mutations significantly impaired the ability of SorT to transfer electrons to SorU, where activities were reduced 17 to 46-fold compared to SorT^WT, precluding determination of kinetic parameters. This was accompanied by the observation of conformational changes in both the introduced Gln and Lys residues in the crystal structure of the enzymes. Taking into account data collected by others on related SOE mutations we propose that the formation and maintenance of an electron transfer complex between the Mo centre and electron accepting heme groups is the main function of the central arginine, and that the reduced turnover and increases in K_Msulfite are caused by the inefficient operation of the oxidative half reaction of the catalytic cycle in enzymes carrying these mutations.     

Purification of a three-subunit ubiquinol-cytochrome c oxidoreductase complex from Paracoccus denitrificans

A ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complex has been purified from the plasma membrane of aerobically grown Paracoccus denitrificans by extraction with dodecyl maltoside and ion exchange chromatography of the extract. The purified complex contains two spectrally and thermodynamically distinct b cytochromes, cytochrome c1, and a Rieske-type iron-sulfur protein. Optical spectra indicate absorption peaks at 553 nm for cytochrome c1 and at 560 and 566 nm for the high and low potential hemes of cytochrome b. The spectrum of cytochrome b560 is shifted to longer wavelength by antimycin. The Paracoccus bc1 complex consists of only three polypeptide subunits. On the basis of their relative electrophoretic mobilities, these have apparent molecular masses of 62, 39, and 20 kDa. The 62- and 39-kDa subunits have been identified as cytochromes c1 and b, respectively. The 20-kDa subunit is assumed to be the Rieske-type iron-sulfur protein on the basis of its molecular weight and the presence of an EPR-detectable signal typical of this iron-sulfur protein in the three-subunit complex. The Paracoccus bc1 complex catalyzes reduction of cytochrome c by ubiquinol with a turnover of 470 s-1. This activity is inhibited by antimycin, myxothiazol, stigmatellin, and hydroxyquinone analogues of ubiquinone, all of which inhibit electron transfer in the cytochrome bc1 complex of the mitochondrial respiratory chain. The electron transfer functions of the Paracoccus complex thus appear to be similar, and possibly identical, to those of the bc1 complex of eukaryotic mitochondria. The Paracoccus bc1 complex has the simplest subunit composition and one of the highest turnover numbers of any bc1 complex isolated from any species to date. These properties suggest that the structural requirements for electron transfer from ubiquinol to cytochrome c are met by a small number of peptides and that the "extra" peptides occurring in the mitochondrial bc1 complexes serve some other function(s), possibly in biogenesis or insertion of the complex into that organelle.     

The 1.25 A resolution structure of the diheme NapB subunit of soluble nitrate reductase reveals a novel cytochrome c fold with a stacked heme arrangement

The diheme cytochrome NapB constitutes the small subunit of a periplasmic nitrate reductase found in a wide variety of bacterial species, including pathogens. The NapB protein is essential in transferring electrons to the large catalytic subunit NapA, which subsequently reduces nitrate to nitrite. Here we present the crystal structure of a proteolyzed form of recombinant NapB from Haemophilus influenzae, which was determined by the multiple-wavelength anomalous dispersion (MAD) method at 1.25 A resolution. This structure shows an unprecedented fold, confirming that NapB proteins belong to a new class of cytochromes. The two heme groups have nearly parallel heme planes and are stacked at van der Waals distances with an iron-to-iron distance of only 9.9 A, two structural features that are also present in the split-Soret diheme cytochrome c from Desulfovibrio desulfuricans ATCC 27774, which is otherwise unrelated in the peptide chain folding pattern. The two propionate side chains on both heme groups are hydrogen-bonded to each other, a structural characteristic that to date also has not been reported in any other heme protein. The propionates of one of the heme groups are pulled toward the interior of the molecule due to a salt bridge and a number of hydrogen bonds between the propionates and conserved residues. We propose a hypothetical but plausible model of the NapAB complex in which the four redox centers are positioned in a virtually linear configuration which spans a distance of nearly 40 A, suggesting an efficient pathway for the transfer of electrons from NapC, the physiological electron donor of NapB, to a nitrate molecule at the catalytic site of NapA.     

The catalytic role of subunit IV of the cytochrome b6-f complex from spinach chloroplast

The catalytic role of subunit IV, the Mr 17,000 protein, in the chloroplast cytochrome b6-f complex was established through trypsinolysis of the complex under controlled conditions. When purified chloroplast cytochrome b6-f complex, 1 mg/ml, in 50 mM Tris-succinate buffer (pH 7.0) containing 1% sodium cholate and 10% glycerol is treated with 80 micrograms of trypsin at room temperature for various lengths of time, the activity of the cytochrome b6-f complex decreases as the incubation time increases. A maximal inactivation of 80% is reached at 7 min of incubation. The trypsin inactivation is accompanied by the destruction of the proton translocation activity of the complex. No alteration of absorption and EPR spectral properties was observed in the trypsin-inactivated complex. Subunit IV is the only subunit in the cytochrome b6-f complex that is digested by trypsin, and the degree of digestion correlates with the decrease of electron transfer activity. The binding of azido-Q to subunit IV of the complex decreases as the extent of inactivation of the cytochrome b6-f complex by trypsin increases. The residue molecular mass of trypsin cleaved subunit IV is about 14 kDa, suggesting that the cleavage site is at lysine 119 or arginine 125 or 126. When the thylakoid membrane was assayed for cytochrome b6-f complex activity, very little activity was observed; and the activity was not sensitive to trypsinolysis. Upon sonication, activity and sensitivity to trypsinolysis was greatly increased, suggesting that subunit IV protrudes from the lumen side of the membrane.     

The formate complex of the cytochrome bo quinol oxidase of Escherichia coli exhibits a 'g = 12' EPR feature analogous to that of 'slow' cytochrome oxidase.

The cytochrome bo quinol oxidase of Escherichia coli is homologous in sequence and in structure to cytochrome aa3 type cytochrome oxidase in subunit I, which contains the catalytic core. The cytochrome bo enzyme forms a formate complex which exhibits 'g = 12' and 'g = 2.9' EPR signals at X band; similar signals have previously been observed only in association with the 'slow' and formate-ligand states of cytochrome oxidase. These signals arise from transitions within integral spin multiples identified with the homologous heme-copper binuclear catalytic centers in both enzymes.     

Effect of subunit IV on superoxide generation by Rhodobacter sphaeroides cytochrome bc1 complex

Previous studies indicate that the three-subunit cytochrome bc₁ core complex of Rhodobacter sphaeroides contains a fraction of the electron transfer activity of the wild-type enzyme. Addition of subunit IV to the core complex increases electron transfer activity to the same level as that of the wild-type complex. This activity increase may result from subunit IV preventing electron leakage, from the low potential electron transfer chain, and reaction with molecular oxygen, producing superoxide anion. This suggestion is based on the following observations: (1) the extent of cytochrome b reduction in the three-subunit core complex, by ubiquinol, in the presence of antimycin A, never reaches the same level as that in the wild-type complex; (2) the core complex produces 4 times as much superoxide anion as does the wild-type complex; and (3) when the core complex is reconstituted with subunit IVs having varying reconstitutive activities, the activity increase in reconstituted complexes correlates with superoxide production decrease and extent of cytochrome b reduction increase.     

Reactivity, secondary structure, and molecular topology of the Escherichia coli sulfite reductase flavodoxin-like domain

The flavodoxin-like domain, missing in the three-dimensional structure of the monomeric, simplified model of the Escherichia coli sulfite reductase flavoprotein component (SiR-FP), has now been expressed independently. This 168 amino acid protein was named SiR-FP18 with respect to its native molecular weight and represents the FMN-binding domain of SiR-FP. This simplified biological object has kept the main characteristics of its counterpart in the native protein. It could incorporate FMN exclusively and stabilize a neutral air-stable semiquinone radical. Both the radical and the fully reduced forms of SiR-FP18 were able to transfer their electrons to DCPIP or cytochrome c quantitatively. SiR-FP18 was able to form a highly stable complex with SiR-HP, the hemoprotein component of the sulfite reductase containing an iron-sulfur cluster coupled to a siroheme. In agreement with the postulated catalytic cycle of SiR-FP, only the fully reduced form of SiR-FP18 could transfer one electron to SiR-HP, the transferred electron being localized exclusively on the heme. As isolated SiR-FP18 has kept the main characteristics of the FMN-binding domain of the native protein, a structural analysis by NMR was performed in order to complete the partial structure obtained previously. Structural modeling was performed using sequence homologues, cytochrome P450 reductase (CPR; 29% identity) and bacterial cytochrome P450 (P450-BM3; 26% identity), as conformational templates. These sequences were anchored using common secondary structural elements identified from heteronuclear NMR data measured on the protein backbone. The resulting structural model was validated, and subsequently refined using residual (C(alpha)-C', N-H(N), and C'-H(N)) dipolar couplings measured in an anisotropic medium. The overall fold of SiR-FP18 is very similar to that of bacterial flavodoxins and of the flavodoxin-like domain in CPR or P450-BM3.     

Suicide inactivation of cytochrome c oxidase: catalytic turnover in the absence of subunit III alters the active site

The catalytic core of cytochrome c oxidase is composed of three subunits: I, II, and III. Subunit III is a highly hydrophobic membrane protein that contains no redox centers; its role in cytochrome oxidase function is not obvious. Here, subunit III has been removed from the three-subunit mitochondrial-like oxidase of Rhodobacter sphaeroides by detergent washing. The resulting two-subunit oxidase, subunit III (-), is highly active. Ligand-binding analyses and resonance Raman spectroscopy show that its heme a(3)-Cu(B) active site is normal. However, subunit III (-) spontaneously and irreversibly inactivates during O(2) reduction. At pH 7.5, its catalytic lifetime is only 2% that of the normal oxidase. This suicide inactivation event primarily alters the active site. Its ability to form specific O(2) reduction intermediates is lost, and CO binding experiments suggest that the access of O(2) to reduced heme a(3) is inhibited. Reduced heme a accumulates in response to a decrease in the redox potential of heme a(3); electron transfer between the hemes is inhibited. Ligand-binding experiments and resonance Raman analysis show that increased flexibility in the structure of the active site accompanies inactivation. Cu(B) is partially lost. It is proposed that suicide inactivation results from the dissociation of a ligand of Cu(B) and that subunit III functions to prevent suicide inactivation by maintaining the structural integrity of the Cu(B) center via long-range interactions.     

Structural and biochemical identification of a novel bacterial oxidoreductase

By using a bioinformatics screen of the Escherichia coli genome for potential molybdenum-containing enzymes, we have identified a novel oxidoreductase conserved in the majority of Gram-negative bacteria. The identified operon encodes for a proposed heterodimer, YedYZ in Escherichia coli, consisting of a soluble catalytic subunit termed YedY, which is likely anchored to the membrane by a heme-containing trans-membrane subunit termed YedZ. YedY is uniquely characterized by the presence of one molybdenum molybdopterin not conjugated by an additional nucleotide, and it represents the only molybdoenzyme isolated from E. coli characterized by the presence of this cofactor form. We have further characterized the catalytic subunit YedY in both the molybdenum- and tungsten-substituted forms by using crystallographic analysis. YedY is very distinct in overall architecture from all known bacterial reductases but does show some similarity with the catalytic domain of the eukaryotic chicken liver sulfite oxidase. However, the strictly conserved residues involved in the metal coordination sphere and in the substrate binding pocket of YedY are strikingly different from that of chicken liver sulfite oxidase, suggesting a catalytic activity more in keeping with a reductase than that of a sulfite oxidase. Preliminary kinetic analysis of YedY with a variety of substrates supports our proposal that YedY and its many orthologues may represent a new type of membrane-associated bacterial reductase.     

Electron transfer through center o of the cytochrome b-c1 complex of yeast mitochondria involves subunit VII, the ubiquinone-binding protein

The role of subunit VII, the ubiquinone-binding protein of the cytochrome b-c1 complex, in electron transfer reactions was investigated in yeast mitochondria. Preincubation of submitochondrial particles with specific antibody against subunit VII prior to addition of either succinate, NADH, or the reduced form of the decyl analogue of ubiquinol resulted in an approximately 40% increase in the extent of cytochrome c1 reduction compared with controls containing preimmune serum. Addition of antimycin, an inhibitor of center i, to submitochondrial particles resulted in a 21% decrease in the rate and a 36% decrease in the extent of cytochrome c1 reduction by succinate. Preincubation of submitochondrial particles with the antibody against subunit VII prior to addition of antimycin resulted in an increase in both the rate and extent of cytochrome c1 reduction to the levels observed in the control without inhibitor. The addition of myxothiazol (an inhibitor of center o), myxothiazol plus antimycin, or alkyl hydroxynaphthoquinone (an inhibitor analogue of ubiquinone) resulted in an almost complete inhibition in both the rate and extent of cytochrome c1 reduction; however, preincubation with the antibody against subunit VII prior to addition of these inhibitors resulted in a significant increase in cytochrome c1 reduction. These results confirm our previous report (Japa, S., Zhu, Q. S., and Beattie, D. S. (1987) J. Biol. Chem. 262, 5441-5444) that subunit VII is involved in electron transfer reactions at center o of the b-c1 complex. We suggest that the binding of antibody to subunit VII inhibits the transfer of electrons to cytochrome b-566. Consequently, two electrons are transferred to the iron-sulfur protein and cytochrome c1 through an antimycin-insensitive pathway. Moreover, the antibody may change the conformation of subunit VII, such that the myxothiazol and hydroxynaphthoquinone binding sites are partially blocked thus permitting electron flow to cytochrome c1.     

Uncovering the molecular mode of action of the antimalarial drug atovaquone using a bacterial system

Atovaquone is an antiparasitic drug that selectively inhibits electron transport through the parasite mitochondrial cytochrome bc1 complex and collapses the mitochondrial membrane potential at concentrations far lower than those at which the mammalian system is affected. Because this molecule represents a new class of antimicrobial agents, we seek a deeper understanding of its mode of action. To that end, we employed site-directed mutagenesis of a bacterial cytochrome b, combined with biophysical and biochemical measurements. A large scale domain movement involving the iron-sulfur protein subunit is required for electron transfer from cytochrome b-bound ubihydroquinone to cytochrome c1 of the cytochrome bc1 complex. Here, we show that atovaquone blocks this domain movement by locking the iron-sulfur subunit in its cytochrome b-binding conformation. Based on our malaria atovaquone resistance data, a series of cytochrome b mutants was produced that were predicted to have either enhanced or reduced sensitivity to atovaquone. Mutations altering the bacterial cytochrome b at its ef loop to more closely resemble Plasmodium cytochrome b increased the sensitivity of the cytochrome bc1 complex to atovaquone. A mutation within the ef loop that is associated with resistant malaria parasites rendered the complex resistant to atovaquone, thereby providing direct proof that the mutation causes atovaquone resistance. This mutation resulted in a 10-fold reduction in the in vitro activity of the cytochrome bc1 complex, suggesting that it may exert a cost on efficiency of the cytochrome bc1 complex.     

Mutational analyses of the photosynthetic reaction center-bound triheme cytochrome subunit and cytochrome c2 in the purple bacterium Rhodovulum sulfidophilum

The purple photosynthetic bacterium Rhodovulum sulfidophilum has an unusual reaction center- (RC-) bound cytochrome subunit with only three hemes, although the subunits of other purple bacteria have four hemes. To understand the electron-transfer pathway through this subunit, three mutants of R. sulfidophilum were constructed and characterized: one lacking the RC-bound cytochrome subunit, another one lacking cytochrome c(2), and another one lacking both of these. The mutant lacking the RC-bound cytochrome subunit was grown photosynthetically with about half the growth rate of the wild type, indicating that the presence of the cytochrome subunit, while not indispensable, is still advantageous for the photosynthetic electron transfer to support its growth. The mutant lacking both the cytochrome subunit and cytochrome c(2) showed a slower rate of growth by photosynthesis (about a fourth of that of the wild type), indicating that cytochrome c(2) is the dominant electron donor to the RC mutationally devoid of the cytochrome subunit. On the other hand, the mutant lacking only the cytochrome c(2) gene grew photosynthetically as fast as the wild type, indicating that cytochrome c(2) is not the predominant donor to the RC-bound triheme cytochrome subunit. We further show that newly isolated soluble cytochrome c-549 with a redox midpoint potential of +238 mV reduced the photooxidized cytochrome subunit in vitro, suggesting that c-549 mediates the cytochrome c(2)-independent electron transfer from the bc(1) complex to the RC-bound cytochrome subunit. These results indicate that the soluble components donating electrons to the RC-bound triheme cytochrome subunit are somewhat different from those of other purple bacteria.     

Kinetic and structural evidence for the importance of Tyr236 for the integrity of the Mo active site in a bacterial sulfite dehydrogenase

The sulfite dehydrogenase from Starkeya novella is the only known sulfite-oxidizing enzyme that forms a permanent heterodimeric complex between a molybdenum and a heme c-containing subunit and can be crystallized in an electron transfer competent conformation. Tyr236 is a highly conserved active site residue in sulfite oxidoreductases and has been shown to interact with a nearby arginine and a molybdenum-oxo ligand that is involved in catalysis. We have created a Tyr236 to Phe substitution in the SorAB sulfite dehydrogenase. The purified SDH(Y236F) protein has been characterized in terms of activity, structure, intramolecular electron transfer, and EPR properties. The substituted protein exhibited reduced turnover rates and substrate affinity as well as an altered reactivity toward molecular oxygen as an electron acceptor. Following reduction by sulfite and unlike SDH(WT), the substituted enzyme was reoxidized quickly in the presence of molecular oxygen, a process reminiscent of the reactions of the sulfite oxidases. SDH(Y236F) also exhibited the pH-dependent CW-EPR signals that are typically observed in vertebrate sulfite oxidases, allowing a direct link of CW-EPR properties to changes caused by a single-amino acid substitution. No quantifiable electron transfer was seen in laser flash photolysis experiments with SDH(Y236F). The crystal structure of SDH(Y236F) clearly shows that as a result of the substitution the hydrogen bonding network surrounding the active site is disturbed, resulting in an increased mobility of the nearby arginine. These disruptions underline the importance of Tyr236 for the integrity of the substrate binding site and the optimal alignment of Arg55, which appears to be necessary for efficient electron transfer.     

Electron transfer by diflavin reductases

Diflavin reductases are enzymes which emerged as a gene fusion of ferredoxin (flavodoxin) reductase and flavodoxin. The enzymes of this family tightly bind two flavin cofactors, FAD and FMN, and catalyze transfer of the reducing equivalents from the two-electron donor NADPH to a variety of one-electron acceptors. Cytochrome P450 reductase (P450R), a flavoprotein subunit of sulfite reductase (SiR), and flavoprotein domains of naturally occurring flavocytochrome fusion enzymes like nitric oxide synthases (NOS) and the fatty acid hydroxylase from Bacillus megaterium are some of the enzymes of this family. In this review the results of the last decade of research are summarized, and some earlier results are reevaluated as well. The kinetic mechanism of cytochrome c reduction is analyzed in light of other results on flavoprotein interactions with nucleotides and cytochromes. The roles of the binding sites of the isoalloxazine rings of the flavin cofactors and conformational changes of the protein in electron transfer are discussed. It is proposed that minor conformational changes during catalysis can potentiate properties of the redox centers during the catalytic turnover. A function of the aromatic residue that shields the isoalloxazine ring of the FAD is also proposed.     

A novel electron transfer mechanism suggested by crystallographic studies of mitochondrial cytochrome bc1 complex.

The crystal structure of bovine mitochondrial cytochrome bc1 complex, an integral membrane protein complex of 11 different subunits with a total molecular mass of 242 kDa, demonstrated a tightly associated dimer consisting of three major regions: a matrix region primarily made of subunits core1, core2, 6, and 9; a transmembrane-helix region of 26 helices in the dimer contributed by cytochrome b, cytochrome c1, the Rieske iron-sulfur protein (ISP), subunits 7, 10, and 11; and an intermembrane-space region composed of extramembrane domains of ISP, cytochrome c1, and subunit 8. The structure also revealed the positions of and distances between irons of prosthetic groups, and two symmetry related cavities in the transmembrane-helix region upon dimerization of the bc1 complex. Extensive crystallographic studies on crystals of bc1 complexed with inhibitors of electron transfer identified binding pockets for both Qo and Qi site inhibitors. Discrete binding sites for subtypes of Qo site inhibitors have been mapped onto the Qo binding pocket, and bindings of different subtypes of Qo site inhibitors are capable of inducing dramatic conformational changes in the extramembrane domain of ISP. A novel electron transfer mechanism for the bc1 complex consistent with crystallographic observations is discussed.     

Wolinella succinogenes quinol:fumarate reductase-2.2-A resolution crystal structure and the E-pathway hypothesis of coupled transmembrane proton and electron transfer

The structure of the respiratory membrane protein complex quinol:fumarate reductase (QFR) from Wolinella succinogenes has been determined by X-ray crystallography at 2.2-A resolution [Nature 402 (1999) 377]. Based on the structure of the three protein subunits A, B, and C and the arrangement of the six prosthetic groups (a covalently bound FAD, three iron-sulfur clusters, and two haem b groups), a pathway of electron transfer from the quinol-oxidising dihaem cytochrome b in the membrane to the site of fumarate reduction in the hydrophilic subunit A has been proposed. The structure of the membrane-integral dihaem cytochrome b reveals that all transmembrane helical segments are tilted with respect to the membrane normal. The "four-helix" dihaem binding motif is very different from other dihaem-binding transmembrane four-helix bundles, such as the "two-helix motif" of the cytochrome bc(1) complex and the "three-helix motif" of the formate dehydrogenase/hydrogenase group. The gamma-hydroxyl group of Ser C141 has an important role in stabilising a kink in transmembrane helix IV. By combining the results from site-directed mutagenesis, functional and electrochemical characterisation, and X-ray crystallography, a residue was identified which was found to be essential for menaquinol oxidation [Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 13051]. The distal location of this residue in the structure indicates that the coupling of the oxidation of menaquinol to the reduction of fumarate in dihaem-containing succinate:quinone oxidoreductases could in principle be associated with the generation of a transmembrane electrochemical potential. However, it is suggested here that in W. succinogenes QFR, this electrogenic effect is counterbalanced by the transfer of two protons via a proton transfer pathway (the "E-pathway") in concert with the transfer of two electrons via the membrane-bound haem groups. According to this "E-pathway hypothesis", the net reaction catalysed by W. succinogenes QFR does not contribute directly to the generation of a transmembrane electrochemical potential.     

Properties and reconstitution of a cytochrome oxidase deficient in subunit III

Three different preparations of beef heart cytochrome oxidase (EC 1.9.3.1) were reconstituted into the membranes of artificial liposomes, and the electrical charge/electron ratios were determined for charge translocation coupled to enzymic activity. Our previously characterised subunit-III-deficient preparation, which apparently lacks H+ translocation capacity [Saraste et al. (1981) Eur. J. Biochem. 115, 261-268] has a decreased charge/electron ratio (0.9-1.0) as determined from the uptake of potassium in the presence of valinomycin, in contrast to the intact reconstituted cytochrome oxidase (1.9-2.0). A third preparation that was depleted of three minor polypeptides by trypsin treatment (these polypeptides are also removed together with subunit III using the present method), but which retains subunit III, had a K+/e- ratio of 1.5 but also a relatively low respiratory control index. The pH-dependence of the Em of cytochrome a determined in the presence of cyanide is abolished in the subunit-III-deficient enzyme. Electron transfer activities are nearly identical for the original and subunit-III-depleted enzymes at an infinite concentration of cytochrome c in a polarographic assay with supplemented phospholipids. The optical spectral properties are very similar for both preparations, but with a small shift to the blue of the alpha-peak in the modified enzyme. These results support the hypothesis that the removal of subunit III abolishes the H+-translocating function of cytochrome oxidase. This occurs by an intrinsic decoupling of H+ transport from electron transfer, and yields a preparation with only half-maximal efficiency of energy conservation.     

The isoforms of yeast cytochrome c oxidase subunit V alter the in vivo kinetic properties of the holoenzyme.

One of the nuclear-coded subunits of yeast cytochrome c oxidase is specified by a gene family composed of two genes, COX5a and COX5b. These genes are regulated differentially by oxygen and encode isoforms of subunit V, designated Va and Vb, which have only 66% primary sequence identity. Yeast cells require one or the other isoform for a functional cytochrome c oxidase (Trueblood, C. E., and Poyton, R. O. (1987) Mol. Cell Biol. 7, 3520-3526). To determine if these isoforms of subunit V alter the catalytic properties of holocytochrome c oxidase, we have analyzed various aspects of cytochrome c oxidase function in intact yeast cells that produce only one type of isoform. From measurements of room temperature turnover numbers and low temperature rates of ligand binding, single turnover cytochrome c oxidation, and internal electron transfer (heme a oxidation), we have found that isozymes which incorporate the Vb isoform have both higher turnover rates and higher rates of heme a oxidation than isozymes which incorporate Va. These findings support the conclusion that the isoforms of subunit V modulate cytochrome c oxidase activity in vivo and suggest that they do so by altering the rates of one or more intramolecular electron transfer reactions.     

Molecular features and mitochondrial import pathway of the 14-kilodalton subunit of cytochrome c reductase from potato.

The cytochrome c reductase complexes from fungi and mammals both contain a 14-kD protein (yeast, 14.4 kD; bovine, 13.4 kD) that does not directly participate in electron transfer but possibly is indirectly involved in the function of the complex and has a role in assembly of the multimeric enzyme. A subunit of comparable size was identified for the bc1 complex of higher plants. The 14-kD protein from potato (Solanum tuberosum) was specifically separated from the isolated protein complex in the presence of 6 M urea and is, therefore, assumed to be a peripheral component. Direct sequence analysis of the proteins from potato and wheat (Triticum aestivum) and isolation of corresponding cDNA clones for the subunit from potato revealed clear similarity to the equivalent proteins from yeast and bovine. The wheat 14-kD protein seems to occur in two isoforms. The 14-kD protein from plants is very hydrophilic, has a characteristic charge distribution, and contains no potential membrane-spanning helices. In vitro import of the radiolabeled 14-kD protein from potato into isolated mitochondria depends on the membrane potential across the inner mitochondrial membrane. The protein seems to lack a cleavable mitochondrial presequence, because it is not processed upon translocation. Possible intramolecular regions involved in targeting of the 14-kD protein to plant mitochondria are discussed.