Structure of the Escherichia coli malate synthase G:pyruvate:acetyl-coenzyme A abortive ternary complex at 1.95 A resolution

Malate synthase, an enzyme of the glyoxylate pathway, catalyzes the condensation and subsequent hydrolysis of acetyl-coenzyme A (acetyl-CoA) and glyoxylate to form malate and CoA. In the present study, we present the 1.95 A-resolution crystal structure of Escherichia coli malate synthase isoform G in complex with magnesium, pyruvate, and acetyl-CoA, and we compare it with previously determined structures of substrate and product complexes. The results reveal how the enzyme recognizes and activates the substrate acetyl-CoA, as well as conformational changes associated with substrate binding, which may be important for catalysis. On the basis of these results and mutagenesis of active site residues, Asp 631 and Arg 338 are proposed to act in concert to form the enolate anion of acetyl-CoA in the rate-limiting step. The highly conserved Cys 617, which is immediately adjacent to the presumed catalytic base Asp 631, appears to be oxidized to cysteine-sulfenic acid. This can explain earlier observations of the susceptibility of the enzyme to inactivation and aggregation upon X-ray irradiation and indicates that cysteine oxidation may play a role in redox regulation of malate synthase activity in vivo. There is mounting evidence that enzymes of the glyoxylate pathway are virulence factors in several pathogenic organisms, notably Mycobacterium tuberculosis and Candida albicans. The results described in this study add insight into the mechanism of catalysis and may be useful for the design of inhibitory compounds as possible antimicrobial agents.     

Crystal structure of Escherichia coli malate synthase G complexed with magnesium and glyoxylate at 2.0 A resolution: mechanistic implications

The crystal structure of selenomethionine-substituted malate synthase G, an 81 kDa monomeric enzyme from Escherichia coli has been determined by MAD phasing, model building, and crystallographic refinement to a resolution of 2.0 A. The crystallographic R factor is 0.177 for 49 242 reflections observed at the incident wavelength of 1.008 A, and the model stereochemistry is satisfactory. The basic fold of the enzyme is that of a beta8/alpha8 (TIM) barrel. The barrel is centrally located, with an N-terminal alpha-helical domain flanking one side. An inserted beta-sheet domain folds against the opposite side of the barrel, and an alpha-helical C-terminal domain forms a plug which caps the active site. Malate synthase catalyzes the condensation of glyoxylate and acetyl-coenzyme A and hydrolysis of the intermediate to yield malate and coenzyme A, requiring Mg(2+). The structure reveals an enzyme-substrate complex with glyoxylate and Mg(2+) which coordinates the aldehyde and carboxylate functions of the substrate. Two strictly conserved residues, Asp631 and Arg338, are proposed to provide concerted acid-base chemistry for the generation of the enol(ate) intermediate of acetyl-coenzyme A, while main-chain hydrogen bonds and bound Mg(2+) polarize glyoxylate in preparation for nucleophilic attack. The catalytic strategy of malate synthase appears to be essentially the same as that of citrate synthase, with the electrophile activated for nucleophilic attack by nearby positive charges and hydrogen bonds, while concerted acid-base catalysis accomplishes the abstraction of a proton from the methyl group of acetyl-coenzyme A. An active site aspartate is, however, the only common feature of these two enzymes, and the active sites of these enzymes are produced by quite different protein folds. Interesting similarities in the overall folds and modes of substrate recognition are discussed in comparisons of malate synthase with pyruvate kinase and pyruvate phosphate dikinase.     

Regulation of Glyoxylate Metabolism in Escherichia coli K-12

The relative contributions of the dicarboxylic acid and the tricarboxylic acid cycles to the oxidative catabolism of glyoxylate in Escherichia coli K-12 were deduced by analysis of mutant strains that were blocked in the formation of glyoxylate carboligase and of malate synthase G (the "glycolate form" of malate synthase). Mutant strains unable to form malate synthase G were unimpaired in their ability to oxidize glyoxylate. Hence, the dicarboxylic acid cycle does not appear to play an essential role in this process. Organisms blocked in the synthesis of glyoxylate carboligase did not oxidize glyoxylate at a detectable rate, indicating that wild-type organisms convert glyoxylate to acetyl-coenzyme A and oxidize it via the tricarboxylic acid cycle. The foregoing evidence indicates that malate synthase G plays an anaplerotic role during growth with glycolate or acetate as the carbon source. The in vivo activity of malate synthase G was not detectable when the intracellular concentration of acetyl-coenzyme A was low, suggesting that this substrate or a closely related metabolite exerts a sensitive positive control over the enzyme. The synthesis of malate synthase G appears to be induced directly by glycolate which may be formed by a constitutive reduced nicotinamide adenine dinucleotide phosphate-dependent glyoxylate reductase in glyoxylate- or acetate-grown cells.     

大肠杆菌苹果酸合酶A的酶学和生理功能研究

苹果酸合酶是乙醛酸循环的关键酶之一。E.coli中苹果酸合酶A(malate synthase A,MSA)由aceB基因编码。根据E.coli基因组序列设计引物,利用PCR技术扩增aceB基因,并将其克隆入pET-29b(+),构建了重组表达质粒pET-MSA。经IPTG诱导,MSA在E.coliRosetta(DE3)中获得高效表达。纯化的MSA蛋白的分子量大小约为60 kDa,最适反应pH值和最适温度分别是pH值8.0、30℃。纯化的蛋白质在Mg2+存在时才能发挥最大的活性,其对乙酰辅酶A的Km和Vmax分别是8.07μM和3.6μM/min。此外构建了MSA和苹果酸合酶G(MSG)基因敲除菌株MG::ΔaceB和MG::ΔaceBΔglcB。研究发现缺少MSA的E.coli突变菌株在乙酸中的生长速率要比野生型菌株慢很多,表明MSA对大肠杆菌在乙酸中的生长起着重要作用。MSG虽然能部分补偿MSA的作用,但是包含MSA的乙醛酸旁路是更有效的乙醛酸代谢途径。     

Identification of residues critical for activity of the wound-induced leucine aminopeptidase (LAP-A) of tomato.

The importance of two putative Zn2+-binding (Asp347, Glu429) and two catalytic (Arg431, Lys354) residues in the tomato leucine aminopeptidase (LAP-A) function was tested. The impact of substitutions at these positions, corresponding to the bovine LAP residues Asp255, Glu334, Arg336, and Lys262, was evaluated in His6-LAP-A fusion proteins expressed in Escherichia coli. Sixty-five percent of the mutant His6-LAP-A proteins were unstable or had complete or partial defects in hexamer assembly or stability. The activity of hexameric His6-LAP-As on Xaa-Leu and Leu-Xaa dipeptides was tested. Most substitutions of Lys354 (a catalytic residue) resulted in His6-LAP-As that cleaved dipeptides at slower rates. The Glu429 mutants (a Zn2+-binding residue) had more diverse phenotypes. Some mutations abolished activity and others retained partial or complete activity. The E429D His6-LAP-A enzyme had Km and kcat values similar to the wild-type His6-LAP-A. One catalytic (Arg431) and one Zn-binding (Asp347) residue were essential for His6-LAP-A activity, as most R431 and D347 mutant His6-LAP-As did not hydrolyze dipeptides. The R431K His6-LAP-A that retained the positive charge had partial activity as reflected in the 4.8-fold decrease in kcat. Surprisingly, while the D347E mutant (that retained a negative charge at position 347) was inactive, the D347R mutant that introduced a positive charge retained partial activity. A model to explain these data is proposed.     

3-D structure of a mutant (Asp101-->Ser) of E.coli alkaline phosphatase with higher catalytic activity.

Mutagenesis of the absolutely conserved residue Asp101 of the non-specific monoesterase alkaline phosphatase (E.C. 3.1.3.1) from E. coli has produced an enzyme with increased kcat. The carboxyl group of the Asp101 residue has been proposed to be involved in the positioning of Arg166 and the formation of the helix that contains the active site Ser102. The crystal structure of the Asp101-->Ser mutant has been refined at 2.5 A to a final crystallographic R-factor of 0.173. The altered active site structure of the mutant is compared with that of the wild-type as well as with the structures of the mutant enzyme soaked in two known alkaline phosphatase inhibitors (inorganic phosphate and arsenate). The changes affect primarily the side chain of Arg166 which, by losing the hydrogen bond interaction with the carboxyl side chain of Asp101, becomes more flexible. This analysis, in conjunction with product inhibition studies of the mutant enzyme, suggests that at high pH (> 7) the enzyme achieves a quicker catalytic turnover by allowing a faster release of the product.     

The crystal structure of E. coli rRNA pseudouridine synthase RluE

Pseudouridine synthase RluE modifies U2457 in a stem of 23 S RNA in Escherichia coli. This modification is located in the peptidyl transferase center of the ribosome. We determined the crystal structures of the C-terminal, catalytic domain of E. coli RluE at 1.2 A resolution and of full-length RluE at 1.6 A resolution. The crystals of the full-length enzyme contain two molecules in the asymmetric unit and in both molecules the N-terminal domain is disordered. The protein has an active site cleft, conserved in all other pseudouridine synthases, that contains invariant Asp and Tyr residues implicated in catalysis. An electropositive surface patch that covers the active site cleft is just wide enough to accommodate an RNA stem. The RNA substrate stem can be docked to this surface such that the catalytic Asp is adjacent to the target base, and a conserved Arg is positioned to help flip the target base out of the stem into the enzyme active site. A flexible RluE specific loop lies close to the conserved region of the stem in the model, and may contribute to substrate specificity. The stem alone is not a good RluE substrate, suggesting RluE makes additional interactions with other regions in the ribosome.     

Role of conserved amino acids in the catalytic activity of Escherichia coli primase

The role of conserved amino acid residues in the polymerase domain of Escherichia coli primase has been studied by mutagenesis. We demonstrate that each of the conserved amino acids Arg146, Arg221, Tyr230, Gly266, and Asp311 is involved in the process of catalysis. Residues Glu265 and Asp309 are also critical because a substitution of each amino acid irreversibly destroys the catalytic activity. Two K229A and M268A mutant primase proteins synthesize only 2-nucleotide products in de novo synthesis reactions under standard conditions. Y267A mutant primase protein synthesizes both full-size and 2-nucleotide RNA, but with no intermediate-size products. From these data we discuss the significant step of the 2-nucleotide primer RNA synthesis by E. coli primase and the role of amino acids Lys229, Tyr267, and Met268 in primase complex stability.     

Overexpression, site-directed mutagenesis, and mechanism of Escherichia coli acid phosphatase.

Site-directed mutagenesis was used to examine the catalytic importance of 2 histidine and 4 arginine residues in Escherichia coli periplasmic acid phosphatase (EcAP). The residues that were selected as targets for mutagenesis were those that were also conserved in a number of high molecular weight acid phosphatases from eukaryotic organisms, including human prostatic and lysosomal acid phosphatases. Both wild type EcAP and mutant proteins were overproduced in E. coli using an expression system based on the T7 RNA polymerase promoter, and the proteins were purified to homogeneity. Examination of the purified mutant proteins by circular dichroism and proton NMR spectroscopy revealed no significant conformational changes. The replacement of Arg16 and His17 residues that were localized in a conserved N-terminal RHGXRXP motif resulted in the complete elimination of EcAP enzymatic activity. Critical roles for Arg20, Arg92, and His303 were also established because the corresponding mutant proteins exhibited residual activities that were not higher than 0.4% of that of wild type enzyme. In contrast, the replacement of Arg63 did not cause a significant alteration of the kinetic parameters. The results are in agreement with a previously postulated distant relationship between acid phosphatases, phosphoglycerate mutases, and fructose-2,6-bisphosphatase. These and earlier results are also consistent with the conclusion that 2 histidine residues participate in the catalytic mechanism of acid phosphatases, with His17 playing the role of a nucleophilic acceptor of the phospho group, whereas His303 may act as a proton donor to the alcohol or phenol.     

Structural basis of UDP-galactose binding by alpha-1,3-galactosyltransferase (alpha3GT): role of negative charge on aspartic acid 316 in structure and activity

alpha-1,3-Galactosyltransferase (alpha3GT) catalyzes the transfer of galactose from UDP-galactose to form an alpha 1-3 link with beta-linked galactosides; it is part of a family of homologous retaining glycosyltransferases that includes the histo-blood group A and B glycosyltransferases, Forssman glycolipid synthase, iGb3 synthase, and some uncharacterized prokaryotic glycosyltransferases. In mammals, the presence or absence of active forms of these enzymes results in antigenic differences between individuals and species that modulate the interplay between the immune system and pathogens. The catalytic mechanism of alpha3GT is controversial, but the structure of an enzyme complex with the donor substrate could illuminate both this and the basis of donor substrate specificity. We report here the structure of the complex of a low-activity mutant alpha3GT with UDP-galactose (UDP-gal) exhibiting a bent configuration stabilized by interactions of the galactose with multiple residues in the enzyme including those in a highly conserved region (His315 to Ser318). Analysis of the properties of mutants containing substitutions for these residues shows that catalytic activity is strongly affected by His315 and Asp316. The negative charge of Asp316 is crucial for catalytic activity, and structural studies of two mutants show that its interaction with Arg202 is needed for an active site structure that facilitates the binding of UDP-gal in a catalytically competent conformation.     

Mutational analysis of conserved outer sphere arginine residues of chalcone synthase

Chalcone synthase (CHS), a key enzyme in flavonoid biosynthesis, catalyses sequential decarboxylative condensations of p-coumaroyl-CoA with three malonyl-CoA molecules and cyclizes the resulting tetraketide intermediate to produce chalcone. Phenylglyoxal, an Arg selective reagent, was found to inactivate the enzyme, although no Arg is found at the active site. Conserved, non-active site Arg residues of CHS were individually mutated and the results were discussed in the context of the 3D structure of CHS. Arg199 and Arg350 were shown to provide important interactions to maintain the structural integrity and foldability of the enzyme. Arg68, Arg172 and Arg328 interact with highly conserved Gln33/Phe215, Glu380 and Asp311/Glu314, respectively, thus helping position the catalytic Cys-His-Asn triad and the (372)GFGPG loop in correct topology at the active site. In particular, a mutation of Arg172 resulted in selective impairment in the cyclization activities of CHS and stilbene synthase, a related enzyme that catalyses a different cyclization of the same tetraketide intermediate. These Arg residues and their interactions are well conserved in other enzymes of the CHS superfamily, suggesting that they may serve similar functions in other enzymes. Mutations of Arg68 and Arg328 had been found in mutant plants that showed impaired CHS activity.     

Mutational analysis of allylic substrate binding site of Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase

Undecaprenyl diphosphate (UPP) synthase catalyzes the sequential cis-condensation of isopentenyl diphosphate (IPP) onto (E,E)-farnesyl diphosphate (FPP). In our previous reports on the Micrococcus luteus B-P 26 UPP synthase, we have shown that the conserved residues in the disordered region from Ser-74 to Val-85 is crucial for the binding of FPP and the catalytic function [Fujikura, K., et al. (2000) J. Biochem. (Tokyo) 128, 917-922] and the existence of a structural P-loop motif for the FPP binding site [Fujihashi, M., et al. (2001) Proc. Natl. Acad. Sci. U.S.A., 98, 4337-4342]. To elucidate the allylic substrate binding site in more detail, we prepared eight mutant enzymes and examined their kinetic behavior. The mutant with respect to the two complementarily conserved Arg residues among the structural P-loop motif, G32R-R42G, retained the activity and showed product distribution pattern exactly similar to that of the wild-type, indicating that the complementarily conserved Arg is important for maintaining the catalytic function. Substitutions of Asp-29, Arg-33, or Arg-80 with Ala resulted in a large loss of enzyme activity, suggesting that these residues are essential for catalytic function. However, the K(m) values of these mutant enzymes for Z-GGPP, which is the first intermediate during the enzymatic cis-condensations of IPP onto FPP, were only moderately different or little changed from those of the wild type. These results suggest that the binding site for the intermediate Z-GGPP having a cis double bond is different to that for the intrinsic allylic substrate, FPP, whose diphosphate moiety is recognized by the structural P-loop.     

Conformational stability of pig citrate synthase and some active-site mutants

The conformational stabilities of native pig citrate synthase (PCS), a recombinant wild-type PCS, and six active-site mutant pig citrate synthases were studied in thermal denaturation experiments by circular dichroism and in urea denaturation experiments by using DTNB to measure the appearance of latent SH groups. His274 and Asp375 are conserved active-site residues in pig citrate synthase that bind to substrates and are implicated in the catalytic mechanism of the enzyme. By site-directed mutagenesis, His274 was replaced with Gly and Arg, while Asp375 was replaced with Gly, Asn, Glu, or Gln. These modifications were previously shown to result in 10(3)-10(4)-fold reductions in enzyme specific activities. The thermal unfolding of pig citrate synthase and the six mutants in the presence and absence of substrates showed large differences in the thermal stabilities of mutant proteins compared to the wild-type pig citrate synthase. The functions of His274 and Asp375 in ligand binding were measured by oxalacetate protection against urea denaturation. These data indicate that active-site mutations that decrease the specific activity of pig citrate synthase also cause an increase in the conformational stability of the protein. These results suggest that specific electrostatic interactions in the active site of citrate synthase are important in the catalytic mechanism in the chemical transformations as well as the conformational flexibility of the protein, both of which are important for the overall catalytic efficiency of the enzyme.     

Studies on site directed mutant pig citrate synthases

The DNAs encoding the non-mutant and mutant forms of pig citrate synthase (PCS) were subcloned into an expression system to determine their synthesis and stability in E. coli gltA- cells that are defective in bacterial citrate synthase. GltA- cells that expressed the non-mutant PCS DNA grew on defined minimal acetate media and produced a constant level of PCS (0.43 U/mg protein). In contrast, when the gltA- cells were transformed with the DNA encoding PCS mutations in His274 or Asp375 the cells did not grow on minimal acetate media. The presence of the mutant PCS proteins in E. coli was confirmed by protein blot and immunoisolation analyses using an antibody specific for porcine heart citrate synthase. The activities of the mutant PCS enzymes were two orders of magnitude less than the non-mutant enzyme in the total cell lysates. The data indicate that the active site amino acids, His274 and Asp375, are essential for the catalysis activity of citrate synthase.     

Functional topology of a surface loop shielding the catalytic center in lipoprotein lipase.

Lipoprotein lipase (LPL), hepatic lipase, and pancreatic lipase show high sequence homology to one another. The crystal structure of pancreatic lipase suggests that it contains a trypsin-like Asp-His-Ser catalytic triad at the active center, which is shielded by a disulfide bridge-bounded surface loop that must be repositioned before the substrate can gain access to the catalytic residues. By sequence alignment, the homologous catalytic triad in LPL corresponds to Asp156-His241-Ser132, absolutely conserved residues, and the homologous surface loop to residues 217-238, a poorly conserved region. To verify these assignments, we expressed in vitro wild-type LPL and mutant LPLs having single amino acid mutations involving residue Asp156 (to His, Ser, Asn, Ala, Glu, or Gly), His241 (to Asn, Ala, Arg, Gln, or Trp), or Ser132 (to Gly, Ala, Thu, or Asp) individually. All 15 mutant LPLs were totally devoid of enzyme activity, while wild-type LPL and other mutant LPLs containing substitutions in other positions were fully active. We further replaced the 22-residue LPL loop which shields the catalytic center either partially (replacing 6 of 22 residues) or completely with the corresponding hepatic lipase loop. The partial loop-replacement chimeric LPL was found to be fully active, and the complete loop-replacement mutant had approximately 60% activity, although the primary sequence of the hepatic lipase loop is quite different. In contrast, replacement with the pancreatic lipase loop completely inactivated the enzyme. Our results are consistent with Asp156-His241-Ser132 being the catalytic triad in lipoprotein lipase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein engineering of uridine phosphorylase from Escherichia coli K-12. II. Comparative study of hybrid and mutant forms of uridine phosphorylases]

Genes for hybrid uridine phosphorylases (UPases) consisting of fragments of amino acid sequences of UPases from Escherichia coli and Salmonella typhimurium were constructed. Producing strains of the corresponding proteins were genetically engineered. Mutant forms of the E. coli K-12 UPase were produced by site-directed mutagenesis. A comparative study of the enzyme properties of the mutant and hybrid forms of bacterial UPases was performed. It was shown that Asp27 unlike Asp5 and Asp29 residues of the E. coli UPase forms part of the active site of the protein. A scheme of the involvement of Asp27 in the binding of inorganic phosphate is proposed.     

Identification of domain-domain docking sites within Clostridium symbiosum pyruvate phosphate dikinase by amino acid replacement

Potential domain-domain docking residues, identified from the x-ray structure of the Clostridium symbiosum apoPPDK, were replaced by site-directed mutagenesis. The steady-state and transient kinetic properties of the mutant enzymes were determined as a way of evaluating docking efficiency. PPDK mutants, in which one of two stringently conserved docking residues located on the N-terminal domain (Arg(219) and Glu(271)) was substituted, displayed largely unimpeded catalysis of the phosphoenolpyruvate partial reaction at the C-terminal domain, but significantly impaired catalysis (>10(4)) of the ATP pyrophosphorylation of His(455) at the N-terminal domain. In contrast, alanine mutants of two potential docking residues located on the N-terminal domain (Ser(262) and Lys(149)), which are not conserved among the PPDKs, exhibited essentially normal catalytic turnover. Arg(219) and Glu(271) were thus proposed to play an important role in guiding the central domain and, hence, the catalytic His(455) into position for catalysis. Substitution of central domain residues Glu(434)/Glu(437) and Thr(453), the respective docking partners of Arg(219) and Glu(271), resulted in mutants impaired in catalysis at the ATP active site. The x-ray crystal structure of the apo-T453A PPDK mutant was determined to test for possible misalignment of residues at the N-terminal domain-central domain interface that might result from loss of the Thr(453)-Glu(271) binding interaction. With the exception of the mutation site, the structure of T453A PPDK was found to be identical to that of the wild-type enzyme. It is hypothesized that the two Glu(271) interfacial binding sites that remain in the T453A PPDK mutant, Thr(453) backbone NH and Met(452) backbone NH, are sufficient to stabilize the native conformation as observed in the crystalline state but may be less effective in populating the reactive conformation in solution.     

Cooperativity in the two-domain arginine kinase from the sea anemone Anthopleura japonicus

cDNAs of the two-domain arginine kinase (AK) (contiguous dimer; denoted by 2D/WT) and its separated domains 1 and 2 (denoted by D1/WT and D2/WT) from the sea anemone Anthopleura japonicus, were cloned into the plasmid pMAL, and recombinant enzymes were expressed in E. coli as MBP fusion proteins. The kinetic parameters kcat, Ka and Kia, were determined for all three AKs. All three enzymes showed distinct AK activity, and had high affinity for arginine (Ka Arg=0.25-0.48 mM). The catalytic efficiency, calculated by kcat/Ka ArgKia ATP, of the 2D/WT enzyme (182 mM(-2)s(-1), the value for one active 40 kDa domain) was two- to three-times higher than values for either D1/WT or D2/WT (80.2 and 86.4mM(-2)s(-1), respectively), suggesting the presence of domain-domain interactions (cooperativity) in the contiguous dimer. The Kia/Ka values of the three enzymes ranged from 0.88 to 1.32, indicating that there is no strong synergism in substrate binding, as seen in typical AKs. Asp62 and Arg193, which are conserved in most AKs and play a key role in stabilizing the substrate-bound structure, are also conserved in the two domains of Anthopleura AK. We replaced Asp62 in D2/WT with Glu or Gly. The catalytic efficiency and Kia/Ka for the D62E mutant were comparable to those of D2/WT, but catalytic efficiency for the D62G mutant was decreased to 13% of that of the D2/WT with a significantly increased value of Kia/Ka (1.92), indicating that Asp62 plays an important role in the expression of AK activity.     

A pair of membrane-embedded acidic residues in the NuoK subunit of Escherichia coli NDH-1, a counterpart of the ND4L subunit of the mitochondrial complex I, are required for high ubiquinone reductase activity

The ND4L subunit of mitochondrial NADH:ubiquinone oxidoreductase (complex I) is an integral membrane protein that contains two highly conserved glutamates within putative trans-membrane helices. We employed complex I from Escherichia coli (NDH-1) to study the role of these residues by site-directed mutagenesis. The conserved glutamates of the NuoK subunit, E36 and E72, were replaced by either Asp or Gln residues, and the effects of the mutations on cell growth and catalysis of electron transfer from deamino-NADH to ubiquinone analogues were examined. Additional mutants that carried acidic residues at selected positions within this domain were also prepared and analyzed. The results indicated that two closely located membrane-embedded acidic residues in NuoK are essential for high rates of ubiquinone reduction, a prerequisite for the growth of cytochrome bo-deficient E. coli cells on malate as the main carbon source. The two acidic residues do not have to be on adjacent helices, and mutual location on the same helix, either helix 2 or 3, at an interval of three amino acids (about one turn of the putative helix), resulted in high activity and good growth phenotypes. Nevertheless, shifting only one of them, either E36 or E72, toward the periplasmic side of the membrane by about one turn of the helix severely hampered activity and growth, whereas moving both acidic residues together to that deeper membrane position stimulated the ubiquinone reductase activity of the enzyme but not cell growth on malate, suggesting impaired energy conservation in this mutant.