Mutagenesis of the L, M, and N subunits of Complex I from Escherichia coli indicates a common role in function

Background

The membrane arm of Complex I (NADH:ubiquinone oxidoreductase) contains three large, and closely related subunits, which are called L, M, and N in E. coli. These subunits are homologous to components of multi-subunit Na⁺/H⁺ antiporters, and so are implicated in proton translocation.

Methodology/Principal Findings

Nineteen site-specific mutations were constructed at two corresponding positions in each of the three subunits. Two positions were selected in each subunit: L_K169, M_K173, N_K158 and L_Q236, M_H241, N_H224. Membrane vesicles were prepared from all of the resulting mutant strains, and were assayed for deamino-NADH oxidase activity, proton translocation, ferricyanide reductase activity, and sensitivity to capsaicin. Corresponding mutations in the three subunits were found to have very similar effects on all activities measured. In addition, the effect of adding exogenous decylubiquinone on these activities was tested. 50 µM decylubiquinone stimulated both deamino-NADH oxidase activity and proton translocation by wild type membrane vesicles, but was inhibitory towards the same activities by membrane vesicles bearing the lysine substitution at the L236/M241/N224 positions.

Conclusions/Significance

The results show a close correlation with reduced activity among the corresponding mutations, and provide evidence that the L, M, and N subunits have a common role in Complex I.     

A mitochondrial DNA variant, identified in Leber hereditary optic neuropathy patients, which extends the amino acid sequence of cytochrome c oxidase subunit I.

A G-to-A transition at nucleotide pair (np) 7444 in the mtDNA was found to correlate with Leber hereditary optic neuropathy (LHON). The mutation eliminates the termination codon of the cytochrome c oxidase subunit I (COI) gene, extending the COI polypeptide by three amino acids. The mutation was discovered as an XbaI restriction-endonuclease-site loss present in 2 (9.1%) of 22 LHON patients who lacked the np 11778 LHON mutation and in 6 (1.1%) of 545 unaffected controls. The mutant polypeptide has an altered mobility on SDS-PAGE, suggesting a structural alteration, and the cytochrome c oxidase enzyme activity of patient lymphocytes is reduced approximately 40% relative to that in controls. These data suggest that the np 7444 mutation results in partial respiratory deficiency and thus contributes to the onset of LHON.     

Leber hereditary optic neuropathy mutations in the ND6 subunit of mitochondrial complex I affect ubiquinone reduction kinetics in a bacterial model of the enzyme

LHON (Leber hereditary optic neuropathy) is a maternally inherited disease that leads to sudden loss of central vision at a young age. There are three common primary LHON mutations, occurring at positions 3460, 11778 and 14484 in the human mtDNA (mitochondrial DNA), leading to amino acid substitutions in mitochondrial complex I subunits ND1, ND4 and ND6 respectively. We have now examined the effects of ND6 mutations on the function of complex I using the homologous NuoJ subunit of Escherichia coli NDH-1 (NADH:quinone oxidoreductase) as a model system. The assembly level of the NDH-1 mutants was assessed using electron transfer from deamino-NADH to the 'shortcut' electron acceptor HAR (hexammine ruthenium), whereas ubiquinone reductase activity was determined using DB (decylubiquinone) as a substrate. Mutant growth in minimal medium with malate as the main carbon source was used for initial screening of the efficiency of energy conservation by NDH-1. The results indicated that NuoJ-M64V, the equivalent of the common LHON mutation in ND6, had a mild effect on E. coli NDH-1 activity, while nearby mutations, particularly NuoJ-Y59F, NuoJ-V65G and NuoJ-M72V, severely impaired the DB reduction rate and cell growth on malate. NuoJ-Met64 and NuoJ-Met72 position mutants lowered the affinity of NDH-1 for DB and explicit C-type inhibitors, whereas NuoJ-Y59C displayed substrate inhibition by oxidized DB. The results are compatible with the notion that the ND6 subunit delineates the binding cavity of ubiquinone substrate, but does not directly take part in the catalytic reaction. How these changes in the enzyme's catalytic properties contribute to LHON pathogenesis is discussed.     

Function of Escherichia coli biotin carboxylase requires catalytic activity of both subunits of the homodimer

Biotin carboxylase catalyzes the ATP-dependent carboxylation of biotin and is one component of the multienzyme complex acetyl-CoA carboxylase that catalyzes the first committed step in fatty acid synthesis. The Escherichia coli biotin carboxylase is readily isolated from the other components of the acetyl-CoA carboxylase complex such that enzymatic activity is retained. The three-dimensional structure of biotin carboxylase, determined by x-ray crystallography, demonstrated that the enzyme is a homodimer consisting of two active sites in which each subunit contains a complete active site. To understand how each subunit contributes to the overall function of biotin carboxylase, we made hybrid molecules in which one subunit had a wild-type active site, and the other subunit contained an active site mutation known to significantly affect the activity of the enzyme. One of the two genes encoded a poly-histidine tag at its N terminus, whereas the other gene had an N-terminal FLAG epitope tag. The two genes were assembled into a mini-operon that was induced to give high level expression of both enzymes. "Hybrid" dimers composed of one subunit with a wild-type active site and a second subunit having a mutant active site were obtained by sequential chromatographic steps on columns of immobilized nickel chelate and anti-FLAG affinity matrices. In vitro kinetic studies of biotin carboxylase dimers in which both subunits were wild type revealed that the presence of the N-terminal tags did not alter the activity of the enzyme. However, kinetic assays of hybrid dimer biotin carboxylase molecules in which one subunit had an active site mutation (R292A, N290A, K238Q, or E288K) and the other subunit had a wild-type active site resulted in 39-, 28-, 94-, and 285-fold decreases in the activity of these enzymes, respectively. The dominant negative effects of these mutant subunits were also detected in vivo by monitoring the rate of fatty acid biosynthesis by [(14)C]acetate labeling of cellular lipids. Expression of the mutant biotin carboxylase genes from an inducible arabinose promoter resulted in a significantly reduced rate of fatty acid synthesis relative to the same strain that expressed the wild type gene. Thus, both the in vitro and in vivo data indicate that both subunits of biotin carboxylase are required for activity and that the two subunits must be in communication during enzyme function.     

The role of transmembrane span 2 in the structure and function of subunit a of the ATP synthase from Escherichia coli

The importance of the second transmembrane span of subunit a of the ATP synthase from Escherichia coli has been established by two approaches. First, biochemical analysis of five cysteine-substitution mutants, four of which were previously constructed for labeling experiments, revealed that only D119C, found within the second transmembrane span, was deleterious to ATP synthase function. This mutant had a greatly reduced growth yield, indicating inefficient ATP synthesis, but it retained a significant level of ATP-driven proton translocation and sensitivity to N,N(')-dicyclohexyl-carbodiimide, indicating more robust function in the direction of ATP hydrolysis. Second, the entire second transmembrane span was probed by alanine-insertion mutagenesis at six different positions, from residues 98 to 122. Insertions at the central four positions from residues 107 to 117 resulted in the inability to grow on succinate minimal medium, although normal levels of membrane-bound ATPase activity and significant levels of subunit a were detected. Double mutants were constructed with a mutation that permits cross-linking to the b subunit. Cross-linked products in the mutant K74C/114iA were seen, indicating no major disruption of the a-b interface due to the insertion at 114. Analysis of the K74C/110iA double mutant indicated that K74C is a partial suppressor of 110iA. In summary, the results support a model in which the amino-terminal, cytoplasmic end of the second transmembrane span has close contact with subunit b, while the carboxy-terminal, periplasmic end is important for proton translocation.     

Conserved lysine residues of the membrane subunit NuoM are involved in energy conversion by the proton-pumping NADH:ubiquinone oxidoreductase (Complex I)

Analysis of the amino acid sequences of subunits NuoM and NuoN in the membrane domain of Complex I revealed a clear common pattern, including two lysines that are predicted to be located within the membrane, and which are important for quinone reductase activity. Site-directed mutations of the amino acid residues E144, K234, K265 and W243 in this pattern were introduced into the chromosomal gene nuoM of Escherichia coli Complex I. The activity of mutated Complex I was studied in both membranes and in purified Complex I. The quinone reductase activity was practically lost in K234A, K234R and E144A, decreased in W243A and K265A but unchanged in E144D. Complex I from all these mutants contained 1 mol tightly bound ubiquinone per mol FMN like wild type enzyme. The mutant enzymes E144D, W243A and K265A had wild type sensitivity to rolliniastatin and complete proton-pumping efficiency of Complex I. Remarkably, the subunits NuoL and NuoH in the membrane domain also appear to contain conserved lysine residues in transmembrane helices, which may give a clue of the mechanism of proton translocation. A tentative principle of proton translocation by Complex I is suggested based on electrostatic interactions of lysines in the membrane subunits.     

An example of Leber hereditary optic neuropathy not involving a mutation in the mitochondrial ND4 gene.

A large Australian family afflicted with Leber's Hereditary Optic Neuropathy (LHON) is analyzed at the nucleotide sequence level in this report. Biochemical assays of platelet mitochondria isolated from members of this family have demonstrated a significant decrease in the specific activity of Complex I (NADH-ubiquinol oxidoreductase) of the electron transport chain. It is shown here, however, that neither this biochemical lesion nor the optic neuropathy are due to the mutation at nucleotide position 11,778 of the mitochondrial ND4 gene first identified by Wallace et al. in several LHON pedigrees. Furthermore, extensive DNA sequencing studies reveal no candidate mutations within the mitochondrial ND3 gene, the ND4L/ND4 genes, or the contiguous tRNA genes. These studies provide the first direct evidence that not all LHON lineages--even those associated with a biochemical defect in mitochondrial respiratory chain Complex I--carry a mutation in the ND4 gene. Members of the Australian LHON family exhibit neurological abnormalities in addition to the well-characterized ophthalmological changes. It is hypothesized that LHON may be a syndrome or set of related diseases in which the clinical abnormalities are a function, at least in part, of the mitochondrial Complex I gene in which the proximate mutation occurs.     

Electron transfer properties of NADH:ubiquinone reductase in the ND1/3460 and the ND4/11778 mutations of the Leber hereditary optic neuroretinopathy (LHON)

We report the electron transfer properties of the NADH:ubiquinone oxidoreductase complex of the respiratory chain (Complex I) in mitochondria of cells derived from LHON patients with two different mutations in mitochondrial DNA (mtDNA). The mutations occur in the mtDNA genes coding for the ND1 and ND4 subunits of Complex I. The ND1/3460 mutation exhibits 80% reduction in rotenone-sensitive and ubiquinone-dependent electron transfer activity, whereas the proximal NADH dehydrogenase activity of the Complex is unaffected. This is in accordance with the proposal that the ND1 subunit interacts with rotenone and ubiquinone. In contrast, the ND4/11778 mutation had no effect on electron transfer activity of the Complex in inner mitochondrial membrane preparations; also Km for NADH and NADH dehydrogenase activity were unaffected. However, in isolated mitochondria with the ND4 mutation, the rate of oxidation of NAD-linked substrates, but not of succinate, was significantly decreased. This suggests that the ND4 subunit might be involved in specific aggregation of NADH-dependent dehydrogenases and Complex I, which may result in fast ('solid state') electron transfer from the former to the latter.     

Cells bearing mutations causing Leber's hereditary optic neuropathy are sensitized to Fas-Induced apoptosis.

Three prevalent mitochondrial DNA pathogenic mutations at positions 11778, 3460, and 14484, which affect different subunits of Complex I, cause retinal ganglion cell death and optic nerve atrophy in Leber's hereditary optic neuropathy (LHON). The cell death is painless and without inflammation, consistent with an apoptotic mechanism. We have investigated the possibility that the LHON mutation confers a pro-apoptotic stimulus and have tested the sensitivity of osteosarcoma-derived cybrid cells carrying the most common and severe mutations (11778 and 3460) to cell death induced by Fas. We observed that LHON cybrids were sensitized to Fas-dependent death. Control cells that bear the same mitochondrial genetic background (the J haplogroup) without the pathogenic 11778 mutation are no more sensitive than other controls, indicating that increased Fas-dependent death in LHON cybrids was induced by the LHON pathogenic mutations. The type of death was apoptotic by several criteria, including induction by Fas, inhibition by the caspase inhibitor zVAD-fmk (zVal-Ala-Asp-fluoro-methyl ketone), activation of DEVDase activity (Asp-Glu-Val-Asp protease), specific cleavage of caspase-3, DNA fragmentation, and increased Annexin-V labeling. These data indicate that the most common and severe LHON pathogenic mutations 11778 and 3460 predispose cells to apoptosis, which may be relevant for the pathophysiology of cell death in LHON, and potential therapy.     

Confirmation of the mitochondrial ND1 gene mutation G3635A as a primary LHON mutation

We report the clinical and genetic characterization of two Chinese LHON families who do not carry the primary LHON-mutations. Mitochondrial genome sequence analysis revealed the presence of a homoplasmic ND1 G3635A mutation in both families. In Family LHON-001, 31 other variants belonging to the East Asian haplogroup R11a were identified and in Family LHON-019, 37 other variants belonging to the East Asian haplogroup D4g were determined. The ND1 G3635A mutation changes the conversed serine¹¹⁰ residue to asparagine. This mutation has been previously described in a single Russian LHON family and has been suggested to contribute to increased LHON expressivity. In addition, a mutation in cytochrome c oxidase subunit II at C7868T (COII/L95F) may act in synergy with G3635A, increasing LHON expressivity in Family LHON-001, which had a higher level of LHON penetrance than Family LHON-019. In summary, the G3635A mutation is confirmed as a rare primary pathogenic mutation for LHON.     

Constraining the Lateral Helix of Respiratory Complex I by Cross-linking Does Not Impair Enzyme Activity or Proton Translocation

Complex I (NADH:ubiquinone oxidoreductase) is a multisubunit, membrane-bound enzyme of the respiratory chain. The energy from NADH oxidation in the peripheral region of the enzyme is used to drive proton translocation across the membrane. One of the integral membrane subunits, nuoL in Escherichia coli, has an unusual lateral helix of ∼75 residues that lies parallel to the membrane surface and has been proposed to play a mechanical role as a piston during proton translocation (Efremov, R. G., Baradaran, R., and Sazanov, L. A. (2010) Nature 465, 441–445). To test this hypothesis we have introduced 11 pairs of cysteine residues into Complex I; in each pair one is in the lateral helix, and the other is in a nearby region of subunit N, M, or L. The double mutants were treated with Cu²⁺ ions or with bi-functional methanethiosulfonate reagents to catalyze cross-link formation in membrane vesicles. The yields of cross-linked products were typically 50–90%, as judged by immunoblotting, but in no case did the activity of Complex I decrease by >10–20%, as indicated by deamino-NADH oxidase activity or rates of proton translocation. In contrast, several pairs of cysteine residues introduced at other interfaces of N:M and M:L subunits led to significant loss of activity, in particular, in the region of residue Glu-144 of subunit M. The results do not support the hypothesis that the lateral helix of subunit L functions like a piston, but rather, they suggest that conformational changes might be transmitted more directly through the functional residues of the proton translocation apparatus.     

A human SCO2 mutation helps define the role of Sco1p in the cytochrome oxidase assembly pathway

Deficiencies in cytochrome oxidase, the terminal enzyme of the mitochondrial respiratory chain, are most often caused by an inability to complete assembly of the enzyme. Pathogenic mutations in SCO2, which encodes a cytochrome oxidase assembly factor, were recently described in several cases of fatal infantile cardioencephalomyopathy. To determine the molecular etiology of these disorders, we describe the generation and characterization of the parallel mutations in the homologous yeast SCO1 gene. We show that the E155K yeast sco1 mutant is respiration-competent, whereas the S240F mutant is not. Interestingly, the S240F mutation allows partial but incorrect assembly of cytochrome oxidase, as judged by an altered cytochrome aa(3) peak. Immunoblot analysis reveals a specific absence of subunit 2 from the cytochrome oxidase in this mutant. Taken together, our data suggest that Sco1p provides copper to the Cu(A) site on subunit 2 at a step occurring late in the assembly pathway. This is the first instance of a yeast cytochrome oxidase assembly mutant that is partially assembled. The S240F mutant also represents a powerful new tool with which to elucidate further steps in the cytochrome oxidase assembly pathway.     

NDUFS4: Creation of a mouse model mimicking a Complex I disorder

The Complex I NADH dehydrogenase–ubiquinone–FeS 4 (NDUFS4) subunit gene is involved in proper Complex I function such that the loss of NDUFS4 decreases Complex I activity resulting in mitochondrial disease. Therefore, a mouse model harboring a point mutation in the NDUFS4 gene was created. An embryonic lethal phenotype was observed in homozygous (NDUFS4^?/?) mutant fetuses. Mitochondrial function was impaired in heterozygous animals based on oxygen consumption, and Complex I activity in NDUFS4 mouse mitochondria. Decreased Complex I activity with unaltered Complex II activity, along with an accumulation of lactate, were consistent with Complex I disorders in this mouse model.     

G204D, a mutation that blocks the proton-conducting D-channel of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides

Cytochrome c oxidase uses the free energy of oxygen reduction to establish a transmembrane proton gradient. The proton-conducting D-channel in this enzyme is the major input pathway for protons which go to the binuclear center for water formation ("chemical protons") and likely the only input pathway for protons that get translocated across the lipid membrane ("pumped protons"). The D-channel starts at an acidic residue near the protein surface (D132, Rhodobacter sphaeroides numbering) and leads to another acidic residue near the binuclear center. Recent studies have shown that mutants that introduce an additional acidic residue in the channel (N139D) have the remarkable effect of accelerating steady-state oxidase activity but completely eliminating proton pumping. In this work, an aspartic acid was introduced at the position of glycine 204, G204D, which is also within the D-channel, and the effects were examined. In contrast to N139D, the G204D mutation results in a dramatic decrease of the steady-state oxygen reductase activity (<2% of wild type) [Aagaard, A., and Brzezinski, P. (2001) FEBS Lett. 494, 157-160]. The residual activity is not coupled to the proton pump, and furthermore, in reconstituted vesicles the mutant enzyme exhibits a reverse respiration control ratio; i.e., the mutant oxidase activity is stimulated rather than inhibited when working against a protonmotive force. Hence, the mutant behaves very much like the D132N, which blocks proton uptake through the D-channel. Single-turnover experiments show that the rate-limiting step in the reaction of O2 with the fully reduced G204D mutant is the F --> O transition, similar to the D132N mutant. The block of the D-channel in the D132N mutant can be partly bypassed by biochemically removing subunit III from the enzyme, indicating that removal of the subunit reveals an alternate entrance for protons to the channel. However, this is not observed with the G204D mutant. This suggests that the cryptic entrance to the D-channel that is revealed by the removal of subunit III is between the levels of G204 and D132.     

Identification of the structural subunits required for formation of the metal centers in subunit I of cytochrome c oxidase of Rhodobacter sphaeroides

Genetic manipulation of the aa(3)-type cytochrome c oxidase of Rhodobacter sphaeroides was used to determine the minimal structural subunit associations required for the assembly of the heme A and copper centers of subunit I. In the absence of the genes for subunits II and III, expression of the gene for subunit I in Rb. sphaeroides allowed purification of a form of free subunit I (subunit I(a)()) that contained a single heme A. No copper was present in this protein, indicating that the heme a(3)-Cu(B) active site was not assembled. In cells expressing the genes for subunits I and II, but not subunit III, two oxidase forms were synthesized that were copurified by histidine affinity chromatography and separated by anion-exchange chromatography. One form was a highly active subunit I-II oxidase containing a full complement of structurally normal metal centers. This shows that association of subunit II with subunit I is required for stable formation of the active site in subunit I. In contrast, subunit III is not required for the formation of any of the metal centers or for the production of an oxidase with wild-type activity. The second product of the cells lacking subunit III was a large amount of a free form of subunit I that appeared identical to subunit I(a)(). Since significant amounts of subunit I(a)() were also isolated from wild-type cells, it is likely that subunit I(a)() will be present in any preparation of the aa(3)-type oxidase isolated via an affinity tag on subunit I.     

The altered activity of complex III may contribute to the high penetrance of Leber's hereditary optic neuropathy in a Chinese family carrying the ND4 G11778A mutation

The ND4 G11778A mutation is the most common mitochondrial DNA mutation leading to Leber's hereditary optic neuropathy (LHON). Despite considerable clinical evidences, the modifier role of nuclear background and mitochondrial haplotypes in phenotypic manifestation of LHON remains poorly understood. We investigated the effect of these modifiers on bioenergetics in lymphoblastoid cell lines derived from five affected subjects of one Chinese family carrying the G11778A mutation and five Chinese controls. Significant reductions in the activities of complexes I and III were observed in mutant cell lines from the Chinese family, whereas the mutant cell lines from other families carrying the same mutation exhibited only reduced activity of complex I. The reduced activities of complexes I and III caused remarkably higher reductions of ATP synthesis in mutant cell lines from the Chinese family than those from other families. The deficient respiration increased generation of reactive oxygen species. The defect in complex III activity, likely resulting from the mitochondrial haplotype or nuclear gene alteration, worsens mitochondrial dysfunction caused by the G11778A mutation, thereby causing extremely high penetrance and expressivity of optic neuropathy in this Chinese family. Our data provide the first experimental evidence that altered activity of complex III modulates the phenotypic manifestation of LHON-associated G11778A mutation. Thus, our findings may provide new insights into the pathophysiology of LHON.     

Enzymatic properties of the membrane-bound NADH oxidase system in the aerobic respiratory chain of Bacillus cereus

Membranes prepared from Bacillus cereus KCTC 3674, grown aerobically on a complex medium, oxidized NADH exclusively, whereas deamino-NADH was little oxidized. The respiratory chain-linked NADH oxidase exhibited an apparent K(m) value of approximately 65 microM for NADH. The maximum activity of the NADH oxidase was obtained at about pH 8.5 in the presence of 0.1 M KCl (or NaCl). Respiratory chain inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) inhibited the activity of the NADH oxidase by about 90% at a concentration of 40 microM. Interestingly, rotenone and capsaicin inhibited the activity of the NADH oxidase by about 60% at a concentration of 40 microM and the activity was also highly sensitive to Ag(+).     

Mutagenesis of an arginine- and lysine-rich domain in the gp91(phox) subunit of the phagocyte NADPH-oxidase flavocytochrome b558

Site-directed mutagenesis was used to generate a series of mutants harboring point or multiple substitutions within the hydrophilic, polybasic domain of gp91(phox) encompassed by residues 86-102, which was previously identified as a site of interaction with p47(phox) during phagocyte NADPH oxidase assembly. Recombinant wild-type or mutant gp91(phox) was expressed in a human myeloid leukemia cell line in which the endogenous gp91(phox) gene was disrupted by gene targeting. NADPH oxidase activity was measured in a cytochrome c reduction assay following granulocytic differentiation of cells that expressed recombinant gp91(phox). Expression of a gp91(phox) mutant in which amino acids 89-97 were replaced with nine alternate amino acids abolished NADPH oxidase activity. Expression of gp91(phox) mutants R89T, D95A, D95R, R96A, R96E, or K102T did not significantly affect NADPH oxidase activity. However, mutations of individual or paired arginine residues at positions 91 and 92 had substantial effects on superoxide generation. The R91E/R92E mutation completely abolished both NADPH oxidase activity and membrane-translocation of the cytosolic oxidase proteins p47(phox), p67(phox), Rac1, and Rac2. The phorbol 12-myristate 13-acetate-induced rate of superoxide production was reduced by approximately 75% in cells expressing R91T/R92A, R91E, or R92E gp91(phox) along with an increased lag time to the maximal rates of superoxide production relative to cells expressing wild-type gp91(phox). Taken together, these results demonstrate that Arg91 and Arg92 of gp91(phox) are essential for flavocytochrome b558 function in granulocytes and suggest that these residues participate in the interaction of gp91(phox) with the cytosolic oxidase proteins.     

Disruption of the nuclear gene encoding the 20.8-kDa subunit of NADH:ubiquinone reductase ofNeurospora mitochondria

The nuclear gene coding for the 20.8-kDa subunit of the membrane arm of respiratory chain NADH:ubiquinone reductase (Complex I) fromNeurospora crassa, nuo-20.8, was localized on linkage group I of the fungal genome. A genomic DNA fragment containing this gene was cloned and a duplication was created in a strain ofN. crassa by transformation. To generate RIP (repeat-induced point) mutations in the duplicated sequence, the transformant was crossed with another strain carrying an auxotrophic marker on chromosome I. To increase the chance of finding an isolate with a non-functionalnuo-20.8 gene, random progeny from the cross were selected against this auxotrophy since RIP of the target gene will only occur in the nucleus carrying the duplication. Among these, we isolated and characterised a mutant strain that lacks the 20.8 kDa mitochondrial protein, indicating that this cysteine-rich polypeptide is not essential. Nevertheless, the absence of the 20.8-kDa subunit prevents the full assembly of complex I. It appears that the peripheral arm and two intermediates of the membrane arm of the enzyme are still formed in the mutant mitochondria. The NADH:ubiquinone reductase activity of sonicated mitochondria from the mutant is rotenone insensitive. Electron microscopy of mutant mitochondria does not reveal any alteration in the structure or numbers of the organelles.     

Dominant role of the cbb3 oxidase in regulation of photosynthesis gene expression through the PrrBA system in Rhodobacter sphaeroides 2.4.1

In this study, the H303A mutant form of the cbb(3) oxidase (H303A oxidase), which has the H303A mutation in its catalytic subunit (CcoN), was purified from Rhodobacter sphaeroides. The H303A oxidase showed the same catalytic activity as did the wild-type form of the oxidase (WT oxidase). The heme contents of the mutant and WT forms of the cbb(3) oxidase were also comparable. However, the puf and puc operons, which are under the control of the PrrBA two-component system, were shown to be derepressed aerobically in the R. sphaeroides strain expressing the H303A oxidase. Since the strain harboring the H303A oxidase exhibited the same cytochrome c oxidase activity as the stain harboring the WT oxidase did, the aerobic derepression of photosynthesis gene expression observed in the H303A mutant appears to be the result of a defective signaling function of the H303A oxidase rather than reflecting any redox changes in the ubiquinone/ubiquinol pool. It was also demonstrated that ubiquinone inhibits not only the autokinase activity of full-length PrrB but also that of the truncated form of PrrB lacking its transmembrane domain, including the proposed quinone binding sequence. These results imply that the suggested ubiquinone binding site within the PrrB transmembrane domain is not necessary for the inhibition of PrrB kinase activity by ubiquinone. Instead, it is probable that signaling through H303 of the CcoN subunit of the cbb(3) oxidase is part of the pathway through which the cbb(3) oxidase affects the relative kinase/phosphatase activity of the membrane-bound PrrB.