Identification of amino acid residues essential for reconstitutive activity of subunit IV of the cytochrome bc1 complex from Rhodobacter sphaeroides

A region of subunit IV comprising residues 77-85 is identified as essential for interaction with the core complex to restore the bc₁ activity (reconstitutive activity). Recombinant subunit IV mutants with single or multiple alanine substitution at this region were generated and characterized to identify the essential amino acid residues. Residues 81-84, with sequence of YRYR, are required for reconstitutive activity of subunit IV, because a mutant with these four residues substituted with alanine has little activity, while a mutant with alanine substitution at residues 77-80 and 85 have the same reconstitutive activity as that of the wild-type IV. The positively charged group at R-82 and R-84 and both the hydroxyl group and aromatic group at Y-81 and Y-83 are essential. The interactions between these four residues of subunit IV and residues of core subunits are also responsible for the stability of the complex. However, these interactions are not essential for the incorporation of subunit IV into the bc₁ complex.     

Subunit IV of cytochrome bc1 complex from Rhodobacter sphaeroides. Localization of regions essential for interaction with the three-subunit core complex

Recombinant subunit IV mutants which identify the regions essential for restoration of bc(1) activity to the three-subunit core complex of Rhodobacter sphaeroides were generated and characterized. Four C-terminal truncated mutants: IV(1-109), IV(1-85), IV(1-76), and IV(1-40) had 100, 0, 0, and 0% of reconstitutive activity of the wild-type IV, indicating that residues 86-109 are essential. IV(1-109) is associated with the core complex in the same manner as the wild-type IV while mutants IV(1-85), IV(1-76), and IV(1-40) do not associate with the core complex, indicating that subunit IV requires its transmembrane helix region (residues 86-109) for assembly into the bc(1) complex. Since GST-IV(86-109) fusion protein has little reconstitutive activity, some region(s) in residues 1-85 are required for bc(1) activity restoration after subunit IV is incorporated into the complex through the transmembrane helix, presumably by interaction with cytochrome b in the core complex. The interacting regions are identified as residues 41-53 and 77-85, since mutants IV(21-109), IV(41-109), IV(54-109), and IV(77-109) had 95, 98, 53, and 53% of the reconstitutive activity of the wild-type IV. These two interacting regions are on the cytoplasmic side of the chromatophore membrane and closed to the DE loop and helix G of cytochrome b, respectively.     

The role of the supernumerary subunit of Rhodobacter sphaeroides cytochrome bc1 complex

The smallest molecular weight subunit (subunit IV), which contains no redox prosthetic group, is the only supernumerary subunit in the four-subunit Rhodobacter sphaeroides bc1 complex. This subunit is involved in Q binding and the structural integrity of the complex. When the cytochrome bc1 complex is photoaffinity labeled with [3H]azido-Q derivative, radioactivity is found in subunits IV and I (cytochrome b), indicating that these two subunits are responsible for Q binding in the complex. When the subunit IV gene (fbcQ) is deleted from the R. sphaeroides chromosome, the resulting strain (RSdeltaIV) requires a period of adaptation before the start of photosynthetic growth. The cytochrome bc1 complex in adapted RSdeltaIV chromatophores is labile to detergent treatment (60-75% inactivation), and shows a four-fold increase in the Km for Q2H2. The first two changes indicate a structural role of subunit IV; the third change supports its Q-binding function. Tryptophan-79 is important for structural and Q-binding functions of subunit IV. Subunit IV is overexpressed in Escherichia coli as a GST fusion protein using the constructed expression vector, pGEX/IV. Purified recombinant subunit IV is functionally active as it can restore the bc1 complex activity from the three-subunit core complex to the same level as that of wild-type or complement complex. Three regions in the subunit IV sequence, residues 86-109, 77-85, and 41-55, are essential for interaction with the core complex because deleting one of these regions yields a subunit completely or partially unable to restore cytochrome bc1 from the core complex.     

The extra fragment of the iron-sulfur protein (residues 96-107) of Rhodobacter sphaeroides cytochrome bc1 complex is required for protein stability

Sequence alignment of the Rieske iron-sulfur protein (ISP) of cytochrome bc(1) complex from various sources reveals that bacterial ISPs contain an extra fragment. To study the role of this fragment in bacterial cytochrome bc(1) complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes with deletion or single- or multiple-alanine substitution at various positions of this fragment (residues 96-107) were generated and characterized. The ISPDelta(96-107), ISP(96-107)A, and ISP(104-107)A mutant cells, in which residues 96-107 of ISP are deleted, and residues 96-107 and 104-107 are substituted with alanine, respectively, do not grow photosynthetically and show no bc(1) complex activity in intracytoplasmic membranes prepared from these mutant cells. The ISP(96-99)A, in which residues 96-99 are substituted with alanine, grows photosynthetically at a rate comparable to that of the complement cells, whereas ISP(100-103)A, in which residues 100-103 are substituted with alanine, has a longer lag period prior to photosynthetic growth. Chromatophores prepared from these two mutant cells have 48% and 9% of the bc(1) activity found in the complement chromatophores. The loss (or decrease) of bc(1) activity in these mutant membranes results from a lack (or decrease) of ISP in the membrane due to ISP protein instability and not from mutations affecting the assembly of cytochromes b and c(1) into the membrane, the binding affinity of cytochrome b to cytochrome c(1), or the ability of these two cytochromes to interact with ISP or subunit IV. The order of essentiality of residues in this fragment is residues 104-107 > residues 100-103 > residues 96-99.     

Reconstitution of mitochondrial processing peptidase from the core proteins (subunits I and II) of bovine heart mitochondrial cytochrome bc(1) complex

Mature core I and core II proteins of the bovine heart mitochondrial cytochrome bc(1) complex were individually overexpressed in Escherichia coli as soluble proteins using the expression vector pET-I and pET-II, respectively. Purified recombinant core I and core II alone show no mitochondrial processing peptidase (MPP) activity. When these two proteins are mixed together, MPP activity is observed. Maximum activity is obtained when the molar ratio of these two core proteins reaches 1. This indicates that only the two core subunits of thebc(1) complex are needed for MPP activity. The properties of reconstituted MPP are similar to those of Triton X-100-activated MPP in the bovine bc(1) complex. When Rieske iron-sulfur protein precursor is used as substrate for reconstituted MPP, the processing activity stops when the amount of product formation (subunit IX) equals the amount of reconstituted MPP used in the system. Addition of Triton X-100 to the product-inhibited reaction mixture restores MPP activity, indicating that Triton X-100 dissociates bound subunit IX from the active site of reconstituted MPP. The aromatic group, rather than the hydroxyl group, at Tyr(57) of core I is essential for reconstitutive activity.     

The role of extra fragment at the C-terminal of cytochrome b (Residues 421-445) in the cytochrome bc1 complex from Rhodobacter sphaeroides

Sequence alignment of cytochrome b of the cytochrome bc1 complex from various sources reveals that bacterial cytochrome b contain an extra fragment at the C terminus. To study the role of this fragment in bacterial cytochrome bc1 complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc1 complexes with progressive deletion from this fragment (residues 421-445) were generated and characterized. The cytbDelta-(433-445) bc1 complex, in which 13 residues from the C-terminal end of this fragment are deleted, has electron transfer activity, subunit composition, and physical properties similar to those of the complement complex, indicating that this region of the extra fragment is not essential. In contrast, the electron transfer activity, binding of cytochrome b, ISP, and subunit IV to cytochrome c1, redox potentials of cytochromes b and c1 in the cytbDelta-(427-445), cytbDelta-(425-445), and cytbDelta-(421-445) mutant complexes, in which 19, 21, or all residues of this fragment are deleted, decrease progressively. EPR spectra of the [2Fe-2S] cluster and the cytochromes b in these three deletion mutant bc1 complexes are also altered; the extent of spectral alteration increases as this extra fragment is shortened. These results indicate that the first 12 residues (residues 421-432) from the N-terminal end of the C-terminal extra fragment of cytochrome b are essential for maintaining structural integrity of the bc1 complex.     

Role of PCDH10 and its hypermethylation in human gastric cancer

Cytochrome bc(1) complex catalyzes the reaction of electron transfer from ubiquinol to cytochrome c (or cytochrome c(2)) and couples this reaction to proton translocation across the membrane. Crystallization of the Rhodobacter sphaeroides bc(1) complex resulted in crystals containing only three core subunits. To mitigate the problem of subunit IV being dissociated from the three-subunit core complex during crystallization, we recently engineered an R. sphaeroides mutant in which the N-terminus of subunit IV was fused to the C-terminus of cytochrome c(1) with a 14-glycine linker between the two fusing subunits, and a 6-histidine tag at the C-terminus of subunit IV (c(1)-14Gly-IV-6His). The purified fusion mutant complex shows higher electron transfer activity, more structural stability, and less superoxide generation as compared to the wild-type enzyme. Preliminary crystallization attempts with this mutant complex yielded crystals containing four subunits and diffracting X-rays to 5.5? resolution.     

Evidence for the head domain movement of the rieske iron-sulfur protein in electron transfer reaction of the cytochrome bc1 complex

The three-dimensional structure of the mitochondrial cytochrome bc1 complex suggests that movement of the extramembrane domain (head) of the Rieske iron-sulfur protein (ISP) may play an important role in electron transfer. Such movement requires flexibility in the neck region of ISP, since the head and transmembrane domains of the protein are rather rigid. To test this hypothesis, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc1 complexes with cysteine substitution at various positions in the ISP neck (residues 39-48) were generated and characterized. The mutants with a single cysteine substitution at Ala42 or Val44 and a double cysteine substitution at Val44 and Ala46 (VQA-CQC) or at Ala42 and Ala46 (ADVQA-CDVQC) have photosynthetic growth rates comparable with that of complement cells. Chromatophore membrane and intracytoplasmic membrane (ICM) prepared from these mutants have cytochrome bc1 complex activity similar to that in the complement membranes, indicating that flexibility of the neck region of ISP was not affected by these cysteine substitutions. Mutants with a double cysteine substitution at Ala42 and Val44 (ADV-CDC) or at Pro40 and Ala42 (PSA-CSC) have a retarded (50%) or no photosynthetic growth rate, respectively. The ADV-CDC or PSA-CSC mutant ICM contains 20 or 0% of the cytochrome bc1 complex activity found in the complement ICM. However, activity can be restored by the treatment with beta-mercaptoethanol (beta-ME). The restored activity is diminished upon removal of beta-ME but is retained if the beta-ME-treated membrane is treated with the sulfhydryl reagent N-ethylmaleimide or p-chloromercuribenzoic acid. These results indicate that the loss of bc1 complex activity in the ADV-CDC or PSA-CSC mutant membranes is due to disulfide bond formation, which increases the rigidity of ISP neck and, in turn, decreases the mobility of the head domain. Using the conditions developed for the isolation of His-tagged complement cytochrome bc1 complex, a two-subunit complex (cytochromes b and c1) is obtained from all of the double cysteine-substituted mutants. This suggests that introduction of two cysteines in the neck region of ISP weakens the interactions between cytochromes b, ISP, and subunit IV.     

Introduction of a non-native disulfide bridge to human lysozyme by cysteine scanning mutagenesis

To examine whether the disulfide bridge between residues 65 and 81 can be replaced by a non-native disulfide bridge in the mutant h-lysozyme C77/95A and whether the formation of such a new disulfide bridge affects the folding of the protein, cysteine scanning mutagenesis has been performed within two discontinuous segments (residues 61-67 for the mutant C65/77/95A, and 74-84 for the mutant C77/81/95A). The position of the Cys residue at 65 or 81 was continuously shifted by site-directed mutagenesis. Of the mutants, only substitution of Cys for Trp64 allowed the secretion of mutant h-lysozyme(W64C) into the medium in a sufficient amount for analysis. After the purification, the mutant enzyme was obtained as two components (W64C-A and W64C-B). The only difference between A and B was that A had a peptide bond cleaved between Ala77 and His78. A non-native disulfide bridge between residues 64-81 was found in both components. Little difference was observed in CD spectra among wild-type and mutant enzymes. It is likely that the tertiary structure of the W64C mutant might be distorted at the location, because the directions of amino acid side chains at positions of 64 and 81 are shown to be opposite to each other in wild-type h-lysozyme by X-ray crystallographic analysis.     

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

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

The role of an extra fragment of cytochrome b (residues 309-326) in the cytochrome bc1 complex from Rhodobacter sphaeroides

In bacterial cytochrome b of the cytochrome bc(1) complex, there is an extra fragment located between the amphipathic helix ef and the transmembrane helix F compared to the mitochondrial counterparts. In this work, mutants at various positions of this extra fragment were generated in Rhodobacter sphaeroides in an effort to investigate its specific role in the bacterial bc(1) complex. The total deletion [cytb-Delta(309-326)] and alanine substitution [cytb-(309-326)A] mutant complexes have about 20% of the bc(1) activity found in the wild-type complex. Mutant complexes of cytb-(309-311)A, cytb-(312-314)A, cytb-(315-317)A, cytb-(318-321)A, cytb-(322-323)A, cytb-(324-326)A, cytb-(F323A), and cytb-(S322A) have respectively 87%, 85%, 89%, 100%, 32%, 90%, 100%, and 32% of the bc(1) activity, indicating that the S322 of cytochrome b is important. EPR spectral analysis reveals that the [2Fe-2S] cluster in the cytb-(S322A) mutant complex has a broadened and shifted g(x)() signal (g = 1.76). The rate of superoxide anion (O(2)(*)(-)) generation is 4 times higher in the cytb-(S322A) mutant complex than in the wild-type or mutant complexes of S322T, S322Y, or S322C. These results support the idea that alanine substitution at S322 of cytochrome b causes conformational changes at the Q(o) site by weakening the binding between cytochrome b and ISP through hydrogen bonding provided by the hydroxyl group of this residue. This change facilitates electron leakage from the Q(o) site for reaction with molecular oxygen to form superoxide anion, thus decreasing bc(1) activity.     

Functional dissection of a cell-division inhibitor, SulA, of Escherichia coli and its negative regulation by Lon

SulA is induced in Escherichia coli by the SOS response and inhibits cell division through interaction with FtsZ. To determine which region of SulA is essential for the inhibition of cell division, we constructed a series of N-terminal and C-terminal deletions of SulA and a series of alanine substitution mutants. Arginine at position 62, leucine at 67, tryptophan at 77 and lysine at 87, in the central region of SulA, were all essential for the inhibitory activity. Residues 3–27 and the C-terminal 21 residues were dispensable for the activity. The mutant protein lacking N-terminal residues 3–47 was inactive, as was that lacking the C-terminal 34 residues. C-terminal deletions of 8 and 21 residues increased the growth-inhibiting activity in lon ⁺ cells, but not in lon ^? cells. The wild-type and mutant SulA proteins were isolated in a form fused to E. coli maltose-binding protein, and tested in vitro for sensitivity to Lon protease. Lon degraded wild-type SulA and a deletion mutant lacking the N-terminal 93 amino acids, but did not degrade the derivative lacking 21 residues at the C-terminus. Futhermore, the wild-type SulA and the N-terminal deletion mutant formed a stable complex with Lon, while the C-terminal deletion did not. MBP fused to the C-terminal 20 residues of SulA formed a stable complex with, but was not degraded by Lon. When LacZ protein was fused at its C-terminus to 8 or 20 amino acid residues from the C-terminal region of SulA the protein was stable in lon ⁺ cells. These results indicate that the C-terminal 20 residues of SulA permit recognition by, and complex formation with, Lon, and are necessary, but not sufficient, for degradation by Lon.     

Electrostatic interactions in the reconstitution of an SH2 domain from constituent peptide fragments

Fragment complementation has been used to delineate the essential recognition elements for stable folding in Src homology 2 (SH2) domains by using NMR spectroscopy, alanine scanning, and surface plasmon resonance. The unfolded 9-kD and 5-kD peptide fragments formed by limited proteolytic digestion of the N-terminal SH2 domain from the p85alpha subunit of phosphatidylinositol 3'-kinase fold into an active native-like structure on interaction with one another. The corresponding 5-kD fragment of the homologous Src protein, however, was not capable of structurally complementing the p85 9-kD fragment, indicating that fragment complementation among these SH2 domains is sensitive to the sequence differences between the Src and p85 domains. Partial complementation and folding activity could be recovered with hybrid sequences of these SH2 domains. Complete alanine scanning of the 5-kD p85 fragment was used to identify the sequence recognition elements required for complex formation. The alanine substitutions in the p85 5-kD fragment that abolished binding affinity with the cognate 9-kD fragment correlate well with highly conserved residues among SH2 domains that are either integrally involved in core packing or found at the interface between fragments. Surprisingly, however, mutation of a nonconserved surface-exposed aspartic acid to alanine was found to have a significant effect on complementation. A single additional mutation of arginine to aspartic acid allowed for recovery of native structure and increased the thermal stability of the designed Src-p85 chimera by 18 degrees C. This modification appears to relieve an unfavorable surface electrostatic interaction, demonstrating the importance of surface charge interactions in protein stability.     

Functional dissection of a cell-division inhibitor, SulA, of Escherichia coli and its negative regulation by Lon

SulA is induced in Escherichia coli by the SOS response and inhibits cell division through interaction with FtsZ. To determine which region of SulA is essential for the inhibition of cell division, we constructed a series of N-terminal and C-terminal deletions of SulA and a series of alanine substitution mutants. Arginine at position 62, leucine at 67, tryptophan at 77 and lysine at 87, in the central region of SulA, were all essential for the inhibitory activity. Residues 3–27 and the C-terminal 21 residues were dispensable for the activity. The mutant protein lacking N-terminal residues 3–47 was inactive, as was that lacking the C-terminal 34 residues. C-terminal deletions of 8 and 21 residues increased the growth-inhibiting activity in lon ⁺ cells, but not in lon ⁻ cells. The wild-type and mutant SulA proteins were isolated in a form fused to E. coli maltose-binding protein, and tested in vitro for sensitivity to Lon protease. Lon degraded wild-type SulA and a deletion mutant lacking the N-terminal 93 amino acids, but did not degrade the derivative lacking 21 residues at the C-terminus. Futhermore, the wild-type SulA and the N-terminal deletion mutant formed a stable complex with Lon, while the C-terminal deletion did not. MBP fused to the C-terminal 20 residues of SulA formed a stable complex with, but was not degraded by Lon. When LacZ protein was fused at its C-terminus to 8 or 20 amino acid residues from the C-terminal region of SulA the protein was stable in lon ⁺ cells. These results indicate that the C-terminal 20 residues of SulA permit recognition by, and complex formation with, Lon, and are necessary, but not sufficient, for degradation by Lon. Received: 8 October 1996 / Accepted: 27 November 1996     

Examination of the functional roles of 5 highly conserved residues in the cytochrome b subunit of the bc1 complex of Rhodobacter sphaeroides.

The cytochrome b subunit of the bc1 complexes contains two cytochrome components (bL and bH) and is the locus of both a quinol-oxidizing site (Qo or Qz) and a quinone-reducing site (Qi or Qc). Sequence alignments of this subunit from over 20 eukaryotic and prokaryotic species have revealed a remarkable degree of conservation, including approximately 20 totally conserved residues. In this paper, site-directed mutagenesis has been used to examine the structural or functional roles of 5 of these highly conserved residues, Gly48, Gln58, Ser102, Phe104, and Pro202, all predicted to be within transmembrane alpha-helical segments. The mutants were made in the bc1 complex of Rhodobacter sphaeroides, a photosynthetic bacterium. The ability to use spectroscopic, electrochemical, and flash-induced kinetic methods allows the mutants to be analyzed for influences both on cytochrome spectra and thermodynamic properties and on the kinetics of specific electron transfer reactions. The results show that none of the 5 residues is absolutely essential. Substitution of aspartate or valine for Gly48 results in the loss of photosynthetic growth. The G48V mutant assembles a bc1 complex, but with modified cytochromes bH and bL, and a dysfunctional quinone reductase (Qc) site; an alanine is tolerated at this position. Possibly, a small residue is important here for heme packing. Gln58 and Ser102 are the only highly conserved polar residues predicted to be within the transmembrane spans, apart from the histidines which are heme axial ligands. Neither Gln58 nor Ser102 is essential for assembly or function of the bc1 complex, although substitution of other amino acids in these positions does cause subtle, but measurable changes. Phe104 lies midway between the axial ligands to cytochromes bL and bH and can be modeled to project in the space separating the two hemes. Replacement of this highly conserved aromatic residue by isoleucine has no measurable influence on the rate of electron transfer through the cytochrome b chain containing the two hemes. Finally, Pro202 is a totally conserved proline which is in the middle of transmembrane helix D, in between the 2 histidines which provide ligands to the hemes. No major inhibition of electron transfer resulted from replacing this proline by a leucine, although subtle changes in spectra of the b cytochromes and their electrochemical properties were noted.     

The Role of the Supernumerary Subunit of Rhodobactersphaeroides Cytochrome bc 1 Complex

The smallest molecular weight subunit (subunit IV), which contains no redox prosthetic group,is the only supernumerary subunit in the four-subunit Rhodobacter sphaeroides bc ₁ complex.This subunit is involved in Q binding and the structural integrity of the complex. When thecytochrome bc ₁ complex is photoaffinity labeled with [³H]azido-Q derivative, radioactivity isfound in subunits IV and I (cytochrome b), indicating that these two subunits are responsiblefor Q binding in the complex. When the subunit IV gene (fbcQ) is deleted from the R.sphaeroides chromosome, the resulting strain (RSIV) requires a period of adaptation beforethe start of photosynthetic growth. The cytochrome bc ₁ complex in adapted RSIVchromatophores is labile to detergent treatment (60–75% inactivation), and shows a four-fold increasein the K _m for Q₂H₂. The first two changes indicate a structural role of subunit IV; the thirdchange supports its Q-binding function. Tryptophan-79 is important for structural andQ-binding functions of subunit IV. Subunit IV is overexpressed in Escherichia coli as a GSTfusion protein using the constructed expression vector, pGEX/IV. Purified recombinant subunitIV is functionally active as it can restore the bc ₁ complex activity from the three-subunit corecomplex to the same level as that of wild-type or complement complex. Three regions in thesubunit IV sequence, residues 86–109, 77–85, and 41–55, are essential for interaction withthe core complex because deleting one of these regions yields a subunit completely or partiallyunable to restore cytochrome bc ₁ from the core complex.     

Structure/function relationships in mitochondrial cytochrome b revealed by the kinetic and circular dichroic properties of two yeast inhibitor-resistant mutants.

The kinetic and circular dichroic properties of two yeast mutants that are resistant towards specific inhibitors of the mitochondrial cytochrome bc1 complex have been characterized. Both of these mutants have an altered cytochrome b gene in which aromatic residues are exchanged with non-polar residues in a highly conserved region of the protein. The mutant resistant to myxothiazol and mucidin that contains the substitution Phe129----Leu is not greatly affected either in its ubiquinol:cytochrome c reductase or in the spectral properties of cytochrome b. On the other hand, the mutant resistant to stigmatellin that contains the substitution Ile147----Phe shows a large decrease of the catalytic efficiency for ubiquinol and of the maximal turnover of its reductase activity. This stigmatellin mutant also shows an altered circular-dichroic spectrum of the low-potential haem of cytochrome b. This study provides biochemical and biophysical information for identifying a region in mitochondrial cytochrome b that may fulfill a crucial role in the binding of ubiquinol to the bc1 complex. The results are discussed also in terms of the structural model of cytochrome b having a core of four transmembrane helices.     

Identification of the active site residues of Pseudomonas aeruginosa protease IV. Importance of enzyme activity in autoprocessing and activation

Protease IV is a lysine-specific endoprotease produced by Pseudomonas aeruginosa whose activity has been correlated with corneal virulence. Comparison of the protease IV amino acid sequence to other bacterial proteases suggested that amino acids His-72, Asp-122, and Ser-198 could form a catalytic triad that is critical for protease IV activity. To test this possibility, site-directed mutations by alanine substitution were introduced into six selected residues including the predicted triad and identical residues located close to the triad. Mutations at any of the amino acids of the predicted catalytic triad or Ser-197 caused a loss of enzymatic activity and absence of the mature form of protease IV. In contrast, mutations at His-116 or Ser-200 resulted in normal processing into the enzymatically active mature form. A purified proenzyme that accumulated in the His-72 mutant was shown in vitro to be susceptible to cleavage by protease IV purified from P. aeruginosa. Furthermore, similarities of protease IV to the lysine-specific endoprotease of Achromobacter lyticus suggested three possible disulfide bonds in protease IV. These results identify the catalytic triad of protease IV, demonstrate that autodigestion is essential for the processing of protease IV into a mature protease, and predict sites essential to enzyme conformation.     

Probing the role of the Fe-S subunit hinge region during Q(o) site catalysis in Rhodobacter capsulatus bc(1) complex

The ubihydroquinone:cytochrome c oxidoreductase, or bc(1) complex, functions according to a mechanism known as the modified Q cycle. Recent crystallographic data have revealed that the extrinsic domain containing the [2Fe2S] cluster of the Fe-S subunit of this enzyme occupies different positions in various crystal forms, suggesting that this subunit may move during ubihydroquinone oxidation. As in these structures the hydrophobic membrane anchor of the Fe-S subunit remains at the same position, the movement of the [2Fe2S] cluster domain would require conformational changes of the hinge region linking its membrane anchor to its extrinsic domain. To probe the role of the hinge region, Rhodobacter capsulatus bc(1) complex was used as a model, and various mutations altering the hinge region amino acid sequence, length, and flexibility were obtained. The effects of these modifications on the bc(1) complex function and assembly were investigated in detail. These studies demonstrated that the nature of the amino acid residues located in the hinge region (positions 43-49) of R. capsulatus Fe-S subunit was not essential per se for the function of the bc(1) complex. Mutants with a shorter hinge (up to five amino acid residues deletion) yielded functional bc(1) complexes, but contained substoichiometric amounts of the Fe-S subunit. Moreover, mutants with increased rigidity or flexibility of the hinge region altered both the function and the assembly or the steady-state stability of the bc(1) complex. In particular, the extrinsic domain of the Fe-S subunit of a mutant containing six proline residues in the hinge region was shown to be locked in a position similar to that seen in the presence of stigmatellin. Interestingly, the latter mutant readily overcomes this functional defect by accumulating an additional mutation which shortens the length of the hinge. These findings indicate that the hinge region of the Fe-S subunit of bacterial bc(1) complexes has a remarkable structural plasticity.     

Subunit gamma of the oxaloacetate decarboxylase Na(+) pump: interaction with other subunits/domains of the complex and binding site for the Zn(2+) metal ion.

The oxaloacetate decarboxylase Na(+) pump of Klebsiella pneumoniae is an enzyme complex composed of the peripheral alpha subunit and the two integral membrane-bound subunits beta and gamma. The alpha subunit consists of the N-terminal carboxyltransferase domain and the C-terminal biotin domain, which are connected by a flexible proline/alanine-rich linker peptide. To probe interactions between the two domains of the alpha subunit and between alpha-subunit domains and the gamma subunit, the relevant polypeptides were synthesized in Escherichia coli and subjected to copurification studies. The two alpha-subunit domains had no distinct affinity toward each other and could, therefore, not be purified as a unit on avidin-sepharose. The two domains reacted together catalytically, however, performing the carboxyl transfer from oxaloacetate to protein-bound biotin. This reaction was enhanced up to 6-fold in the presence of the Zn(2+)-containing gamma subunit. On the basis of copurification with different tagged proteins, the C-terminal biotin domain but not the N-terminal carboxyltransferase domain of the alpha subunit formed a strong complex with the gamma subunit. Upon the mutation of gamma H78 to alanine, the binding affinity to subunit alpha was lost, indicating that this amino acid may be essential for formation of the oxaloacetate decarboxylase enzyme complex. The binding residues for the Zn(2+) metal ion were identified by site-directed and deletion mutagenesis. In the gamma D62A or gamma H77A mutant, the Zn(2+) content of the decarboxylase decreased to 35% or 10% of the wild-type enzyme, respectively. Less than 5% of the Zn(2+) present in the wild-type enzyme was found if the two C-terminal gamma-subunit residues H82 and P83 were deleted. Corresponding with the reduced Zn(2+) contents in these mutants, the oxaloacetate decarboxylase activities were diminished. These results indicate that aspartate 62, histidine 77, and histidine 82 of the gamma subunit are ligands for the catalytically important Zn(2+) metal ion.