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Human Molecular Genetics Advance Access originally published online on July 18, 2006
Human Molecular Genetics 2006 15(17):2543-2552; doi:10.1093/hmg/ddl176
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The MELAS mutations 3946 and 3949 perturb the critical structure in a conserved loop of the ND1 subunit of mitochondrial complex I

Marko Kervinen1, Reetta Hinttala2,4, Heli M. Helander3, Sari Kurki5, Johanna Uusimaa3, Moshe Finel5,6, Kari Majamaa2,7 and Ilmo E. Hassinen1,*

1 Department of Medical Biochemistry and Molecular Biology, 2 Department of Neurology and 3 Department of Pediatrics, University of Oulu, PO Box 5000, Fin-90014, Oulu, Finland, 4 Clinical Research Center, Oulu University Hospital, Fin-90029, Oulu, Finland, 5 Department of Medical Chemistry, University of Helsinki, Fin-00014, Helsinki, Finland, 6 Drug Discovery and Development Technology Center, Faculty of Pharmacy, University of Helsinki, Fin-00014, Helsinki, Finland and 7 Department of Neurology, University of Turku, Fin-20014, Turku, Finland

* To whom correspondence should be addressed. Tel: +358 85375802; Fax: +358 85375811; Email: ilmo.hassinen{at}oulu.fi

Received April 27, 2006; Revised June 14, 2006; Accepted July 7, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
The ND1 subunit gene of the mitochondrial NADH-ubiquinone oxidoreductase (complex I) is a hot spot for mutations causing Leber hereditary optic neuropathy and several mutations causing the mitochondrial encephalopathy, lactic acidosis and stroke-like episodes syndrome (MELAS). We have used Escherichia coli and Paracoccus denitrificans as model systems to study the effect of mutations 3946 and 3949, which change conserved residues in ND1 and cause MELAS. The vicinity of these mutations was also explored with a series of mutations in charged residues. The 3946 mutation results in E214K substitution in human ND1. Replacement of the equivalent residue in E. coli with lysine or glutamine detracted from enzyme assembly and the assembled enzyme was inactive. However, the equivalent E234Q mutant enzyme in P. denitrificans failed to assemble completely (or was rapidly degraded). Also the corresponding substitution with aspartate decreased the enzyme activity in P. denitrificans and E. coli. The 3949-equivalent substitution, Y229H in E. coli, lowered the catalytic activity by 30%. In addition, an activation of the enzyme during catalytic turnover was seen in this bacterial NDH-1, something that was even more pronounced in another mutant in the same loop, D213E. Several other mutations in this region decreased the enzyme activity. The studied MELAS mutations are situated in a matrix-side loop, which appears to be highly sensitive to structural perturbations. The results provide new information on the function of the region affected by the MELAS mutations 3946 and 3949 that is not obtainable from patient samples or current eukaryote models.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
The proton-pumping NADH–ubiquinone oxidoreductase (also called complex I in mitochondria or NDH-1 in bacteria) is a large membrane enzyme consisting of 46 dissimilar subunits in mammalian mitochondria (1,2) and 13 to 14 in bacteria (3–6). Seven of the human complex I subunits, ND1-6 and ND4L, are encoded by the mitochondrial genome (mtDNA) and the rest are transcribed in the cell nucleus and transported into the mitochondria. Complex I has an L-shaped outline with one arm embedded in the mitochondrial inner membrane or bacterial cytoplasmic membrane and the other protruding into the mitochondrial matrix or the bacterial cytoplasm. Complex I catalyzes electron transfer from NADH to ubiquinone, which is the first step in the respiratory chain, and uses the energy released from NADH oxidation to pump four protons across the mitochondrial inner membrane or the bacterial cytoplasmic membrane (7–9). There are several human diseases associated with this enzyme, for a review see Triepels et al. (10).

The NADH binding site and all the known redox cofactors of complex I are located in its peripheral domain and are arranged in a serial fashion, ending in the iron–sulfur cluster N2, which is thought to serve as the electron donor for ubiquinone (11–13). The mechanism of function of this large and important enzyme is poorly understood at present and its high-resolution structure is still not known, although several hypotheses have been put forward over the years [for a review see (14)]. On the basis of the pH-dependence of its midpoint potential, it has been proposed that the iron–sulfur cluster N2 may be part of a direct proton pump (14–16), whereas observations on the existence of several semiquinone species point to a mechanism employing a reverse Q-cycle (14,17–19), and the redox-dependence of its cross-linking pattern and susceptibility to tryptic digestion argue in favor of a conformationally coupled proton pump (20–22).

The mitochondrial genome is a DNA circle of 16.5 kb, containing information for two rRNAs, 22 tRNAs and 13 polypeptides (23). The mutation rate of mtDNA is about an order of magnitude higher than that of nuclear DNA (24,25), and pathogenic mtDNA mutations primarily have a deleterious effect on the turnover capacity of the respiratory chain or on the ability of mitochondria to produce ATP, which in turn can cause severe but variable symptoms. More than 100 pathogenic mtDNA mutations have been reported to date (Mitomap, www.mitomap.org, April 2006). In addition to screening disease-causing mutations in patients with mitochondrial symptoms, the analysis of 2000 complete mtDNA sequences from cohorts all over the world has provided information on the variability of mtDNA (23,26–30).

Mitochondrial diseases display a variety of clinical symptoms ranging from a retinal ganglion cell defect in Leber hereditary optic neuropathy (LHON) to severe neurological syndromes, as in the mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome, where patients suffer from episodic vomiting, seizures and recurrent episodes resembling strokes, with hemiparesis, hemianopsia or cortical blindness (31,32). A 3243A>G-mutation in the mitochondrial tRNALeu(UUR) gene has been found in 80% of MELAS cases, and the majority of the remaining cases result from other mutations in mitochondrial tRNA genes (33). In addition, several mutations in the ND subunit genes of mitochondrial complex I, namely in ND5 (34), ND6 (35) and ND1 (36), have been found to cause MELAS. Interestingly, subunits ND6 and ND1 have also been found to be hot spots for LHON mutations (37,38) and the pathogenic amino acid substitution in these two diseases reside close to each other or even affect the same residue (see Fig. 1 and the references therein). Why the apparently similar mutations cause very different clinical phenotypes is a question that is waiting for an answer.


Figure 1761
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  		Figure 1. Schematic presentation of the membrane topology of the E. coli NuoH subunit. The positions of human disease-associated mutations and the corresponding amino acid changes (human ND1 numbering) are shown. The LHON/IE-L285P patient also harbored the 14484 common LHON mutation. The residues mutated in this work are also highlighted (E. coli NuoH numbering). IE, infantile encephalopathy. The human mutations have been published in (36,38,64–67).

 
A single amino acid change in a respiratory enzyme can cause a mitochondrial disease with devastating symptoms. The pathogenesis of mitochondrial diseases remains largely obscure, especially those caused by mutations affecting complex I. Relatively few such pathogenic mtDNA mutations have been characterized in detail for their effects on NADH oxidation and ubiquinone reduction. This study was set up to evaluate the effect of two MELAS mutations situated in the third matrix-side loop of subunit ND1 on the catalytic properties of the enzyme and the role of a conserved segment in this loop structure (Fig. 2), motivated by a previous observation about changes in ubiquinone binding in the E212Q mutant of Paracoccus denitrificans (39). Because genetic manipulation of mitochondrial DNA is extensively laborious owing to the different genetic code and the location within the mitochondrion, we used a bacterium, the phylogenetic progenitor of a mitochondrion, as a model system. This enables manipulation of a single amino acid in the enzyme and the introduction of deleterious mutations. This bacterial model system was employed here to expand our knowledge of the biochemical features of the mitochondrial disease pathology involved in MELAS and to enable comparison and assessment of the biochemical effects in different diseases.


Figure 1762
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  		Figure 2. Sequence alignment of the third inside loop segment of complex I subunit ND1/NuoH/Nqo8. Residues belonging to the inside ends of the predicted transmembrane helices 5 and 6 are underlined in the E. coli sequence, conserved amino acids are highlighted and the residues mutated in this work are marked with x. Accession numbers for sequences are (in order of appearance): NP 416785.1, P29920, AAA97945.1, P03886, P03887, NP 007562.1, NP 006955.1, CAC28089.2, CAA60353.1, Q37556 and YP 271938.1. The alignment was carried out using Clustal W 1.82 with default parameters.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Deletion and in trans complementation of the nuoH gene
Deletion of the nuoH gene from the Escherichia coli (GV102 strain) genome results in a malate growth-negative phenotype, as previously described for the nuoJK double deletion strain (40). The NDH-1-dependent activities, namely (deamino-NADH) d-NADH:DB (decylubiquinone) oxidoreductase, d-NADH oxidation and d-NADH:HAR (hexammineruthenium) oxidoreductase were minimal (Table 1 and Fig. 3). The d-NADH:DB oxidoreductase represents the full NDH-1 activity, whereas d-NADH oxidation represents the entire respiratory chain, which, in the case of strain GV102, involves NDH-1 and the bd oxidase. The d-NADH:HAR oxidoreductase activity represents a partial NDH-1 activity that is catalyzed by the NADH dehydrogenase domain only. The latter activity was used to estimate the level of NDH-1 assembly.


Figure 1763
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  		Figure 3. Assembly-normalized NDH-1 activities in membrane samples from various mutants. The bars represent the DB reductase/HAR reductase activity ratio in two (mutants) or five (control) independent membrane samples. The values are means±SD. A statistically significant (P<0.05) decline in DB/HAR activity is marked with an asterisk. The HAR reductase activities used for normalization are presented in Table 1.

 


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  		Table 1. Respiratory chain activity in membrane preparations from NuoH mutants and their growth on malate

 
Wild-type nuoH genes, additionally modified with restriction sites at both ends, were cloned into the pETBlue-2 expression plasmid. Site-directed mutagenesis was performed in this control plasmid. The restriction endonuclease site construction resulted in S2G and Q325G substitutions in the gene product. In trans complementation with the control plasmid restores the NDH-1-dependent activities and positive malate growth phenotype (Table 1 and Fig. 3), even though the assembly level is lower than in the parent strain (HAR reductase 1109±229 nmol min–1 mg–1 in the parent strain, GV102). The lower assembly level is probably a result of the non-synchronized expression and assembly of the many NDH-1 subunits from the nuo-operon and the mutated or control nuoH from the complementation plasmid. The effect of the S2G and Q325G on assembly is probably negligible due to the low conservation in these regions. Hence, the results obtained with the different mutants were compared with the control complementation rather than with the parent strain before deletion. This was justified, as the control NDH-1 activity was similar to that of the parent strain GV102 in terms of DB and inhibitor affinities and maximal turnover rate, normalized to the expression level indicated by the d-NADH:HAR oxidoreductase activity (Table 2). It may be added that the assembly level was found to vary somewhat between different membrane preparations of either the control or any given mutant, leading to high variability in specific activities (Table 1). Nevertheless, significantly lower variability was found when the d-NADH:DB oxidoreduction activities were normalized on the basis of the d-NADH:HAR oxidoreductase to obtain an assembly-normalized activity that depends only on the properties of the NDH-1 holoenzyme turnover (Fig. 3).


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  		Table 2. Enzymological characterization of the d-NADH:decylubiquinone reductase activity in NuoH mutants

 
All the mutant subunits in E. coli considered here were assembled to the bacterial membrane, as indicated by the increased HAR reductase activity relative to the HK18 deletion strain (P<0.05 for each of the mutants). The method used is not optimized for the detection of mildly decreased assembly levels because of the mutation, and thus minor instability or assembly failure might remain unnoticed. Nevertheless, instability (or poor assembly) of the enzyme should result in markedly decreased HAR reductase activity, representing the NADH dehydrogenase domain (see also the discussion below).

MELAS mutations
The human MELAS mutations 3946/ND1-E214K and 3949/ND1-Y215H were generated in E. coli, where the homologous replacements are E228K and Y229H in the NuoH subunit of NDH-1, respectively. Both mutant enzymes were assembled on the membrane, as indicated by the increased HAR reductase activities relative to the deletion strain. Interestingly, the mutations in the NuoH-E228 position did not abolish assembly, but with the exception of the conservative Glu to Asp mutation, they did yield the lowest HAR reductase activities of all the mutants that we prepared (Table 1). The NuoH-E228K mutant did not catalyze d-NADH oxidase or DB reductase activities (Table 1 and Fig. 3) and all its colonies displayed a negative malate growth phenotype (Table 1).

In order to define better the effect of the NuoH-E228K mutation, we introduced two additional mutations into this position. The NuoH-E228Q mutation, which removes the acidic group in an otherwise conservative manner, gave rise to an inactive enzyme (Fig. 3), whereas the conservative mutation, NuoH-E228D, which only shortens the amino acid side chain, reduced the activity by 50%.

We also studied the effect of mutations equivalent to E. coli NuoH-E228Q and NuoH-E228D in the NDH-1 of P. denitrificans, a bacterium that is evolutionarily closer to the mammalian mitochondria than E. coli. The latter similarity is also reflected in high sequence homology between its NDH-1 subunits and the corresponding complex I subunits (6). The equivalent residue in P. denitrificans Nqo8 (the ND1 homologue in this bacterium), E234, was replaced with glutamine or aspartate in the same genomic complementation system that was previously used to study the bacterial equivalent of the LHON mutation (41) and the role of other conserved Glu residues (39). The results of this mutagenesis experiment revealed that replacing Nqo8-E234 with Asp did not cause any major changes in NDH-1 activity and that the bacteria were able to grow without the ‘assistance’ of NDH-2 (Table 3). In contrast, none of the Nqo8-E234Q colonies was able to grow in the absence of NDH-2, and they also exhibited negligible d-NADH:Q1 activity (Table 3). Moreover, mutant E234Q, contrary to most other mutants produced in the previous study of P. denitrificans NDH-1 (39), had a very low HAR reductase activity (Table 3), which was in line with the low HAR reductase in E. coli E228Q, although the assembly failure was more evident in the P. denitrificans enzyme. The latter results are in full agreement with the suggestion that an acidic residue in this position is important for the folding of the subunit or assembly of the large enzyme (or else the lack of it leads to rapid degradation).


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  		Table 3. Effects of mutations in the MELAS-3946/E214K position of the Nqo8 subunit of the P. denitrificans on NDH-1 enzyme activity and dependence of growth on NDH-2

 
The second MELAS mutation, 3949/ND1-Y215H, appeared less severe, as the normalized d-NADH oxidase activity and the DB reductase activity in the equivalent E. coli mutant NuoH-Y229H were above 70% of the control. In addition, most colonies of the NuoH-Y229H mutant displayed positive malate growth. Nevertheless, examination of the NDH-1 turnover rate of this mutant revealed an autoactivation phenomenon during its reaction with DB (Fig. 4, trace 4).


Figure 1764
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  		Figure 4. Activation of d-NADH:DB reductase during enzyme turnover. NDH-1 activity increases within the first 2 min of d-NADH-DB oxidoreduction in the NuoH-D213E and NuoH-Y229H mutants. The straight dotted lines represent the initial activities. Membrane preparations from: 1, control; 2, NuoH-D213E; 3, NuoH-D213N; 4, NuoH-Y229H. The initial DB concentration was 100 µM.

 
Mutations in the first half of the third inside loop
There is a conserved glutamate at the end of the fifth transmembrane helix of the NuoH subunit in all NDH-1 enzymes except those of E. coli, Salmonella typhimurium and Klebsiella pneumoniae (Fig. 2). The E. coli residue in this position, Val, has previously been introduced into the P. denitrificans enzyme (39), and we constructed the opposite mutation here, making a NuoH-V206E mutant of the E. coli enzyme. This change was well tolerated, as the assembly-normalized DB reductase activity was reduced only by 10% when compared with the control (Fig. 3), and the Km for DB remained unchanged (Table 2). The NuoH-R209, NuoH-H210, NuoH-D213N and NuoH-E216A mutants displayed reduced assembly normalized DB reductase activities (28–43% decreases relative to the control, see Fig. 3). Selected mutants were analyzed further, and unchanged affinity for DB and reduced maximal turnover velocities were found (Table 2). None of the mutants analyzed here exhibited substrate inhibition, and their d-NADH:DB oxidoreductase activity was 73–84% sensitive to inhibition with 400 µM N-vanillylnonanamide (VNA) in all preparations (NuoH-E228K and NuoH-E228Q not analyzed due to low activity) except for the HK18 deletion strain, where the small amount of residual activity was essentially insensitive to VNA.

Interestingly, the NuoH-D213E mutant enzyme displayed marked activation for the first 2 min of the DB reductase reaction (see Fig. 4, trace 2), i.e. the activity increased significantly during the assay and then remained linear. The activation could not be fully eliminated by pre-incubation of the enzyme in the presence of DB (and in the presence of KCN) for up to 15 min before the d-NADH addition. The activation decreased somewhat when the enzyme was turned over initially with 20 µM d-NADH followed immediately after its depletion by the conventional addition of 80 µM d-NADH and activity measurement. Furthermore, the activation was also seen when the d-NADH oxidase activity of the NuoH-D213E-mutant, but not that of the control NDH-1, was assayed with oxygen as the electron acceptor. It thus appears that the activation phenomenon is an intrinsic catalytic property of the mutant enzyme rather than arising from inadequate experimental conditions and it does not depend on the structure of the hydrophobic tail in ubiquinone.

Growth phenotype in mutants
When the malate growth phenotype was analyzed in E. coli strains containing the mutant NDH-1 enzyme the NuoH-V206E mutant enzyme displayed a positive growth phenotype, whereas all the other mutants with substitutions in the third inside loop region exhibited an intermediate growth phenotype at best. The NuoH-E228K and NuoH-E228Q mutants had a negative growth phenotype, in accordance with the negligible NDH-1 activity. Interestingly, all the NuoH-E216A mutant colonies displayed negative growth, even though the normalized NDH-1 and d-NADH oxidation activities were similar to or higher than those in the NuoH-V206E mutant that displayed positive growth.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
The first mutation observed to lead to mitochondrially inherited disease was the LHON mutation 11778, in 1988 (42). Although the spectrum of well-defined clinical syndromes with known defects in mitochondrial genes is ever-expanding, the relation between the molecular target of the mutation and the clinical phenotype is still obscure. Also, the biochemical effects of the mutations on the respiratory chain enzymes and the consequences for the mitochondrial and further cellular metabolism are still largely unknown. We set out to examine the enzymological effects of selected pathological mitochondrial complex I mutations, focusing on the MELAS mutations in the ND1 subunit (this work) and the LHON mutations in the ND6 subunit (Pätsi, Kervinen, Finel and Hassinen, manuscript in preparation).

The MELAS syndrome is probably the most common maternally inherited mitochondrial disease (43,44). It is known that an apparent complex I deficiency is seen in patient samples with the common 3243A>G-mutation in tRNALeu (45–47), despite the fact that this mutation should affect all mitochondrially transcribed proteins and thus impair each of the respiratory chain complexes I, III, IV and V. The effects of 3243A>G have been studied in hybrid cells, where reduced mitochondrial protein synthesis and particularly reduced complex I activity has been found (48,49). King et al. (48) have hypothesized that the disproportional complex I deficiency results from inaccurately processed mRNA, resulting in an N-terminally truncated ND1 subunit (the tRNALeu gene resides next to the ND1 gene), but this could not be verified by mRNA analysis (49). The role of complex I in MELAS pathogenesis is emphasized by the fact that several mutations in mitochondrially encoded complex I subunit genes cause MELAS (34–36) and an apparent hot spot for MELAS mutations was found in the ND1 subunit gene (36). It is intriguing that this subunit is also a hot spot for LHON mutations and that these mutations all reside in the conserved matrix-side loops (Fig. 1).

The ND1 subunit (the homologues of which are called NuoH or Nqo8 in bacteria, NdhA in the chloroplast or NAD1 in plant mitochondrion) contains eight trans-membrane helices, and the topology of the loops protruding from the membrane has been determined by fusion protein technology (50). The two MELAS mutations studied in this work are located within a loop region that is highly conserved. Furthermore, the residues affected by these mutations are almost fully conserved among the proton pumping NADH–ubiquinone oxidoreductases, including human complex I, E. coli and P. denitrificans NDH-1, enabling modeling of the human mutations in the bacterial enzyme.

The elimination of the acidic group equivalent to the E. coli NuoH-E228 in the bacterial NDH-1 from P. denitrificans (the Nqo8-E234Q mutant) resulted in negligible assembly of this NDH-1 enzyme, and the assembly level was also significantly lower in the NuoH-E228Q and NuoH-E228K mutants in the E. coli case (Table 1), but still higher than in P. denitrificans. The mutation equivalent to NuoH-E228K in the human ND1 subunit causes MELAS (36), and it has been shown that the level of assembly of complex I is significantly reduced in fibroblast mitochondria isolated from a patient carrying this mutation. The complex II-normalized complex I assembly (11% of control) and activity (14%) were approximately similar (36). It should be noted that the patient fibroblasts were heteroplasmic for the mutation in question, with ~45% mutant mtDNA, strongly suggesting that the complex I activity and the assembled complex I enzyme were the products of the unaffected gene copies. Taken together, it now seems that the results for the three distinct enzymes indicate that an acidic residue in this position is needed for proper assembly of the enzyme.

Why does E. coli appear to be less sensitive to the substitution of E228 with a non-acidic residue? Based on the current information and in the absence of a high-resolution structure for this region, we can come up with two possible explanations. The first is a structural deviation in the E. coli enzyme in this region. If the negative charge of E228 is important in folding of the loop or interaction of the loop with other subunits, the additional aspartate residue in this loop in the E. coli enzyme (NuoH-D223) that is lacking from the human and P. denitrificans enzymes, could preserve the charge interaction with the surrounding protein. The second explanation, or a supporting feature for the first hypothesis, is over-expression of the NuoH subunit. This may simply provide more material, or more alternative conformations for the assembly process. If there are variations in loop structure between the expressed subunit molecules, docking with other subunits in a certain favorable conformation might drive the assembly process more efficiently when there are more NuoH subunit ‘candidates’ for docking, i.e. an over-expression situation.

Regardless of the possible assembly defect, both MELAS amino acid substitutions reduced the complex I turnover rate markedly in the E. coli enzyme (Table 1). The effects on the bacterial enzyme were similar to those in patient muscle samples, as the NuoH-E228K-substituted enzyme was found to be practically inactive and NuoH-Y229H was affected to a lesser degree. The ubiquinone reduction kinetics of the NuoH-E228K mutant could not be investigated further because of the low activity. Interestingly, replacement of NuoH-E228 with aspartate, a highly conservative change, also reduced the NDH-1 activity by 50%, but without any significant changes in the DB or VNA binding affinities (Table 2).

Replacement of tyrosine-229 with histidine brought about an activation phenomenon during initial turnover of the enzyme, previously undetected in bacterial complex I homologues. The affinity for DB in the NuoH-Y229H mutant was not reduced and the defect in enzymatic activity resulted from lowered maximal turnover capacity (Vmax).

In order to characterize better the possible effects of the MELAS mutations, we generated a series of mutations in their vicinity, particularly in the highly conserved part of the N-terminal segment of the same inside-facing loop (Fig. 1). All the mutants in this loop segment displayed an ~30–40% decrease in assembly-normalized ubiquinone reductase activity (Table 1). The latter trend also included the threonine and phenylalanine substitutions of the non-conserved NuoH-H210. It thus appears that this region is highly sensitive to structural perturbations.

The largest change in enzyme function among the N-terminal segment mutants was seen in the NuoH-D213E mutant (Table 2). In addition, this mutant displayed remarkable autoactivation at the beginning of the ubiquinone reductase reaction (Fig. 4, trace 2) but still a 36% decline in activity remains to be compared with control. Interestingly, when the original aspartate in this position was replaced by asparagine rather than by glutamate no autoactivation was detected and the decrease in normalized activity was slightly less than in the NuoH-D213E mutant (Table 2). It thus appears that the length of the side chain in position 213 is very important, as the addition of a –CH2– group to the side chain of the original aspartate (D213E) results in a larger change in activity than the elimination of acidity (D213N). It could be speculated that lengthening of the side chain caused steric congestion within the protein, affecting the enzyme activity. Nevertheless, this could be partially overcome by rearrangement during enzyme turnover (activation). The catalytically favorable structure is rapidly lost after cessation of the catalytic reaction, however, as indicated by the reappearance of autoactivation (at least partially) immediately after the end of the reaction. At present, it is too early to say whether this has something to do with the active/inactive transition observed in mitochondrial complex I [see a review in (51)].

Mutations in the ND1 were previously shown to alter the affinity of complex I for ubiquinone and rotenone slightly in human mitochondria (52–54) or in bacteria (39,41). The ND1 subunit has also been reported to be labeled with [3H]dihydrorotenone (55) and [14C]DCCD (56). The iron–sulfur cluster N2-containing PSST subunit (57) is considered to contribute to a ubiquinone binding site together with the 49 kDa subunit (58). The ND1 subunit has been later identified as the low-affinity binding site for a photoaffinity derivative of pyridaben, with functional coupling to the PSST binding site (59). These observations point to a close proximity of the ND1 subunit to the ubiquinone reduction site.

We analyzed the ubiquinone reductase activity of selected mutants in order to find out whether ubiquinone or inhibitor binding is affected by the mutations introduced. All the mutations analyzed resulted in lowered assembly normalized Vmax values (Vmax/HAR) relative to the control and none gave significantly increased Km values, indicating that this region is important for a high turnover capacity in the enzyme but is not directly involved in ubiquinone binding. This is supported by the unchanged affinities and sensitivities for VNA, a C-type complex I inhibitor (60). The activation phenomenon may imply that this loop region has (or can have) some mobility, enabling re-organization of the loop into an active conformation during turnover or binding of the substrates. It is therefore tempting to speculate that this glutamate-rich loop may be involved in the conformation change associated with proton pumping (20–22). In contrast, contrary to the initial hypothesis, ubiquinone binding was found to be unaffected in the mutants involved in the present investigation. If the ubiquinone binding site resides at the interface of subunits 49 kDa and ND1, as originally proposed by Darrouzet et al. (61) [noting the PSST subunit involvement acknowledged later on in Prieur et al. (58)], the studied residues in the third matrix-side loop of the ND1 subunit will not be much involved.

The MELAS mutations 3946 and 3949 affecting the subunit ND1 of complex I decreased the amount of assembled enzyme in the bacterial models and thus probably have a parallel effect on the cellular metabolism as the MELAS mutations in tRNA genes. However, the presence of assembled complex I that is incapable of re-oxidation by ubiquinone, as found in the case of E. coli NuoH-E228K mutant, might have pathological consequences for cell metabolism also in human pathological conditions via increased radical oxygen species generation or the promotion of apoptosis. In addition, the characterization of the new mutants, their partial activity and the newly discovered phenomenon of autoactivation in certain mutants, may provide new tools for studying this large and important enzyme, particularly the involvement of the loop segment studied here in both the assembly of complex I and its activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Materials
DB, NADH, deamino-NADH, DL-malate, VNA and soy bean phospholipids were from Sigma. HAR was purchased from Aldrich, HEPES and MES from AppliChem and KCN and phenylmethylsulfonylfluoride (PMSF) from Merck. Oligonucleotides were purchased from Sigma-Genosys. The QuikChange XL mutagenesis kit was from Stratagene and Phusion DNA polymerase from Finnzymes.

NuoH deletion
A deletion was introduced into the GV102 strain of E. coli (62) according to the procedure described in (40). The oligonucleotides are described in the Supplementary Material, Table S1 with numbers corresponding to those used in table 2 and figure 2 of Kervinen et al. (40), with the exception that UF was initially cloned using the RE-sites SmaI–EcoRI. The flanking regions were sequenced to confirm that no unwanted mutations had been introduced during the procedure. The deletion construct modifies the original stop codon of the nuoG gene and results in a fusion peptide with a 38 amino acid C-terminal extension. The resulting deletion strain was named HK18 and used for expression of the wild-type or mutant nuoH gene (see below).

NuoH expression construct and mutagenesis
Expression plasmids for in trans complementation were prepared by amplifying the nuoH fragment from genomic DNA using primers 9 and 10 (Supplementary Material, Table S1) and subcloning into pETBlue-2 digested with NcoI plus EcoRI, generating the expression plasmid pHwt with a native stop codon. The sequence of the insert in pHwt was verified by DNA sequencing. Point mutations were introduced into pHwt using the QuikChange XL mutagenesis kit (Stratagene) according to the manufacturer's instructions or the Phusion DNA polymerase (Finnzymes), followed by DNA sequencing of the mutant gene. The oligonucleotides used in mutagenesis are listed in Supplementary Material, Table S1.

Bacterial growth and membrane preparation
E. coli bacteria were grown and prepared according to Kervinen et al. (40), with the following modifications. LB was supplemented with 1% arabinose when the transformed deletion strain bacteria were grown for the glycerol stock, and streptomycin was routinely replaced by kanamycin (except for the deletion strain selection). For cell collection, A600 limits of 0.4–0.7 were used and 50 mM Tris–HCl, pH 7.5, 250 mM KCl, 2.5 mM EDTA, 0.2 mM PMSF were used for the cell washing step. The bacteria were broken with a French press in 10 mM MES/KOH, pH 6.0, 10% glycerol (v/v), 5 mM MgCl2, 0.15 mg · mL–1 DTT, 0.2 mM PMSF, 9 µg · mL–1 DNAse I and the membranes were resuspended in the same buffer without DTT to about 1 mL · g–1 of initial cell weight. The protein concentration was determined according to Lowry et al. (63) or with a BCA kit (Pierce).

P. denitrificans genetic manipulation and sample preparation were done as described previously (39).

Enzyme activity measurements
The activity assays were performed using a Shimadzu UV-3000 or Aminco-DW2 dual-wavelength spectrophotometer according to Kervinen et al. (40), with the following exceptions. DB activity assays were performed within 2 weeks of collection of the bacterial sample with 0.1 mg · mL–1 of membrane sample protein, 0.5 mg · mL–1 soy bean phospholipid, 80–95 µM deamino-NADH and 30–400 µM DB (initial concentrations). Activities for DB concentrations between 2 and 30 µM were determined from the reaction progression curve obtained with an initial DB concentration of 30–40 µM. Assays for VNA inhibition kinetics were performed as for the DB assays above, except that an equal volume of VNA (final concentrations 0–400 µM) or ethanol was added before DB. An amount of 0.01 mg · mL–1 of membrane protein and 400 µM VNA were used in the HAR reductase activity measurements.

Statistical analysis
The DB reductase activities for the membrane preparations, comprising 20–40 separate measurements with an even distribution of DB concentrations between 2 and 400 µM, were fitted to Michaelis–Menten one site saturation kinetics using the SigmaPlot 9 program. The cumulative error in the activity measurements performed on the different control membrane preparations was similar to the error calculated when the individual values obtained from the different membrane preparations were compared with each other, indicating that the bacterial growth and membrane preparation procedures did not induce any greater variability into the results than the activity measurements themselves. However, because it was considered more appropriate, the results were evaluated in terms of the differences between the membrane preparations (including the variation in bacterial growth, membrane preparation and activity measurement) where applicable. The statistical significances of differences in enzyme activities were evaluated using the Bonferroni modification of the t-test.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We are indebted to Mrs Aila Holappa and Mrs Raija Pietilä for their skilful technical assistance. This investigation was supported by the Academy of Finland Council for Health and the Sigrid Juselius Foundation. M.K. also acknowledges support from the Evald and Hilda Nissi Foundation and the Finnish Eye Foundation (Silmäsäätiö). The work of R.H. was supported by the Alma and K.A. Snellman Foundation, Oulu, Finland, and the Päivikki and Sakari Sohlberg Foundation. The support of the Arvo and Lea Ylppö Foundation and the Foundation for Pediatric Research for J.U. is also acknowledged.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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