Tuning of the optical and electrochemical properties of the primary donor bacteriochlorophylls in the reaction centre from Rhodobacter sphaeroides: spectroscopy and structure

A series of mutations have been introduced at residue 168 of the L-subunit of the reaction centre from Rhodobacter sphaeroides. In the wild-type reaction centre, residue His L168 donates a strong hydrogen bond to the acetyl carbonyl group of one of the pair of bacteriochlorophylls (BChl) that constitutes the primary donor of electrons. Mutation of His L168 to Phe or Leu causes a large decrease in the mid-point redox potential of the primary electron donor, consistent with removal of this strong hydrogen bond. Mutations to Lys, Asp and Arg cause smaller decreases in redox potential, indicative of the presence of weak hydrogen bond and/or an electrostatic effect of the polar residue. A spectroscopic analysis of the mutant complexes suggests that replacement of the wild-type His residue causes a decrease in the strength of the coupling between the two primary donor bacteriochlorophylls. The X-ray crystal structure of the mutant in which His L168 has been replaced by Phe (HL168F) was determined to a resolution of 2.5 A, and the structural model of the HL168F mutant was compared with that of the wild-type complex. The mutation causes a shift in the position of the primary donor bacteriochlorophyll that is adjacent to residue L168, and also affects the conformation of the acetyl carbonyl group of this bacteriochlorophyll. This conformational change constitutes an approximately 27 degrees through-plane rotation, rather than the large into-plane rotation that has been widely discussed in the context of the HL168F mutation. The possible structural basis of the altered spectroscopic properties of the HL168F mutant reaction centre is discussed, as is the relevance of the X-ray crystal structure of the HL168F mutant to the possible structures of the remaining mutant complexes.     

Structural consequences of the replacement of glycine M203 with aspartic acid in the reaction center from Rhodobacter sphaeroides

Reaction centers with the double mutation Phe M197 to Arg and Gly M203 to Asp (FM197R/GM203D) have been crystallized from an antenna-deficient strain of Rhodobacter sphaeroides, and the structure has been determined at 2.7 A resolution. Unlike in reaction centers with a single FM197R mutation, the Arg M197 residue in the FM197R/GM203D reaction center adopts a position similar to that of the native Phe residue in the wild-type reaction center. Asp M203 is packed in such a way that the gamma-carboxy group interacts with the backbone carbonyl of Arg M197. The Asp M203 residue takes up part of the volume that is occupied in the wild-type reaction center by a water molecule. This water has been proposed to form a hydrogen bond interaction with the 9-keto carbonyl group of the active branch accessory bacteriochlorophyll, particularly when the primary donor bacteriochlorophylls are oxidized. The GM203D mutation therefore appears to remove the possibility of this hydrogen bond interaction by exclusion of this water molecule, as well as altering the local dielectric environment of the 9-keto carbonyl group. We examine whether the observed structural changes can provide new or alternative explanations for the absorbance and electron-transfer properties of reaction centers with the FM197R and GM203D mutations.     

Structural basis of the drastically increased initial electron transfer rate in the reaction center from a Rhodopseudomonas viridis mutant described at 2.00-A resolution

It has previously been shown that replacement of the residue His L168 with Phe (HL168F) in the Rhodopseudomonas viridis reaction center (RC) leads to an unprecedented drastic acceleration of the initial electron transfer rate. Here we describe the determination of the x-ray crystal structure at 2.00-A resolution of the HL168F RC. The electron density maps confirm that a hydrogen bond from the protein to the special pair is removed by this mutation. Compared with the wild-type RC, the acceptor of this hydrogen bond, the ring I acetyl group of the "special pair" bacteriochlorophyll, D(L), is rotated, and its acetyl oxygen is found 1.1 A closer to the bacteriochlorophyll-Mg(2+) of the other special pair bacteriochlorophyll, D(M). The rotation of this acetyl group and the increased interaction between the D(L) ring I acetyl oxygen and the D(M)-Mg(2+) provide the structural basis for the previously observed 80-mV decrease in the D(+)/D redox potential and the drastically increased rate of initial electron transfer to the accessory bacteriochlorophyll, B(A). The high quality of the electron density maps also allowed a reliable discussion of the mode of binding of the triazine herbicide terbutryn at the binding site of the secondary quinone, Q(B).     

Dependence of the bi-functional nature of a sialyltransferase from Neisseria meningitidis on a single amino acid substitution

The L1 immunotype strain 126E of Neisseria meningitidis has been shown to have an N-acetyl-neuraminic acid-containing lipooligosaccharide in which an alpha-linked galactose from a P(k) epitope is substituted at the O6 position (Wakarchuk, W. W., Gilbert, M., Martin, A., Wu, Y., Brisson, J. R., Thibault, P., and Richards, J. C. (1998) Eur. J. Biochem. 254, 626-633). Using a synthetic P(k)-epitope containing acceptor in glycosyltransferase reactions, we were able to show by NMR analysis of the reaction product that the 126E(L1)-derived sialyltransferase can make both alpha-2,3 and alpha-2,6 linkages to the terminal galactose. Gene disruption experiments showed that the lst gene in 126E(L1) was responsible for the in vivo addition of the alpha-2,6-linked N-acetyl-neuraminic acid residue. By site-directed mutagenesis it was possible to change the MC58(L3)-derived enzyme into a bifunctional enzyme with a single amino acid change at position 168, where a glycine was changed to an isoleucine. We performed a gene replacement experiment where the 126E(L1) alpha-2,3/6-sialyltransferase was replaced by allelic exchange with the monofunctional MC58(L3) alpha-2,3-sialyltransferase and with the mutant MC58(L3) allele G168I. We observed that the level of LOS sialylation with the G168I allele was very similar to that of the wild type 126E(L1), indicating that residue 168 is the critical residue for the alpha-2,6-sialyltransferase activity in vitro as well as in vivo.     

Modeling the bacterial photosynthetic reaction center 3: interpretation of effects of site-directed mutagenesis on the special-pair midpoint potential

Interpretation of changes in midpoint potential of the "special pair" in bacterial photosynthetic reaction centers caused by site-directed mutagenesis is discussed in terms of a simple tight-binding model which relates them to concomitant variations in spin distribution between the two bacteriochlorophyll molecules of the special pair. Our analysis improves on previous similar ones by Allen and co-workers [Artz, K., Williams, J. C., Allen, J. P., Lendzian, F., Rautter, J., and Lubitz, W. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 13582; Ivancich, A., Artz, K., Williams, J. C., Allen, J. P., and Mattioli, T. A. (1998) Biochemistry 37, 11812] in that it is both more complete, including electron-phonon coupling, and more accurate. It is applied to analyze data for a series of M160 mutants of Rhodobacter sphaeroides, yielding a value of 0.18+/-0.03 eV for the electronic coupling energy between the highest occupied levels of the two bacteriochlorophylls in the wild-type and a value of the energy offset E(o) between the highest occupied molecular orbitals of the L and M bacteriochlorophylls of 0.14+/-0.03 eV. For a mutant in which the electron hole in the special pair cation is located entirely on the reactive (L) side, a potential of 641+/-30 mV with respect to the normal hydrogen electrode is predicted. This agrees well with the average value ca. 650 mV observed for the heterodimer mutant HL(M202) in which the bacteriochlorophyll on the unreactive M side has been replaced by a bacteriopheophytin, causing extensive charge localization. However, the deduced coupling is found to be very sensitive to small changes in the assumptions used in the model, and various important chemical effects remain to be included.     

Selective enhancement of resonance Raman spectra of separate bacteriopheophytins in Rb. sphaeroides reaction centers

Tunable dye laser excitation of carefully prepared samples of Rb. sphaeroides reaction centers provides richly detailed resonance Raman (RR) spectra of the bacteriopheophytins, H, and the accessory bacteriochlorophylls, B. These spectra demonstrate selective enhancement of the separate bacteriopheophytins on the active (H(L)) and inactive (H(M)) sides of the reaction centers. The spectra are assigned with the aid of normal coordinate analyses using force fields previously developed for porphyrins and reduced porphyrins. Comparison of the H(L) and H(M) vibrational mode frequencies reveals evidence for greater polarization of the acetyl substituent in H(L) than H(M). This polarization is expected to make H(L) easier to reduce, thereby contributing to the directionality of electron transfer from the special pair, P. In addition, the acetyl polarization of H(L) is increased at low temperature (100 K), helping to account for the increase in electron transfer rate. The polarizing field is suggested to arise from the Mg(2+) of the neighboring accessory bacteriochlorophyll, which is 4.9 A from the acetyl O atom. The 100 K spectra show sharpening and intensification of a number of RR bands, suggesting a narrowing of the conformational distribution of chromophores, which is consistent with the reported narrowing of the distribution in electron transfer rates. Excitation at 800 nm produces high-quality RR spectra of the accessory bacteriochlorophylls, and the spectral pattern is unaltered on tuning the excitation to 810 nm in resonance with the upper exciton transition of P. Either the resonance enhancement of P is weak, or the bacteriochlorophyll RR spectra are indistinguishable for P and B.     

Site-specific receptor methylation of FrzCD in Myxococcus xanthus is controlled by a tetra-trico peptide repeat (TPR) containing regulatory domain of the FrzF methyltransferase

Myxococcus xanthus is a gliding bacterium with a complex life cycle that includes swarming, predation and fruiting body formation. Directed movements in M. xanthus are regulated by the Frz chemosensory system, which controls cell reversals. The Frz pathway requires the activity of FrzCD, a cytoplasmic methyl-accepting chemotaxis protein, and FrzF, a methyltransferase (CheR) containing an additional domain with three tetra trico-peptide repeats (TPRs). To investigate the role of the TPRs in FrzCD methylation, we used full-length FrzF and FrzF lacking its TPRs (FrzF^CheR) to methylate FrzCD in vitro . FrzF methylated FrzCD on a single residue, E182, while FrzF^CheR methylated FrzCD on three residues, E168, E175 and E182, indicating that the TPRs regulate site-specific methylation. E168 and E182 were predicted consensus methylation sites, but E175 is methylated on an HE pair. To determine the roles of these sites in vivo , we substituted each methylatable glutamate with either an aspartate or an alanine residue and determined the impact of the point mutants on single cell reversals, swarming and fruiting body formation. Single, double and triple methylation site mutants revealed that each site played a unique role in M. xanthus behaviour and that the pattern of receptor methylation determined receptor activity. This work also shows that methylation can both activate and inactivate the receptor.     

Effects of mutations of active site residues and amino acids interacting with the Omega loop on substrate activation of butyrylcholinesterase

The peripheral anionic site (PAS) of human butyrylcholinesterase is involved in the mechanism of substrate activation by positively charged substrates and ligands. Two substrate binding loci, D70 in the PAS and W82 in the active site, are connected by the Omega loop. To determine whether the Omega loop plays a role in the signal transduction between the PAS and the active site, residues involved in stabilization of the loop, N83, K339 and W430, were mutated. Mutations N83A and N83Q caused loss of substrate activation, suggesting that N83 which interacts with the D70 backbone may be an element of the transducing system. The K339M and W430A mutant enzymes retained substrate activation. Residues W82, E197, and A328 in the active site gorge have been reported to be involved in substrate activation. At butyrylthiocholine concentrations greater then 2 mM, W82A showed apparent substrate activation. Mutations E197Q and E197G strongly reduced substrate activation, while mutation E197D caused a moderate effect, suggesting that the carboxylate of residue E197 is involved in substrate activation. Mutations A328F and A328Y showed no substrate activation, whereas A328G retained substrate activation. Substrate activation can result from an allosteric effect due to binding of the second substrate molecule on the PAS. Mutation W430A was of special interest because this residue hydrogen bonds to W82 and Y332. W430A had strongly reduced affinity for tetramethylammonium. The bimolecular rate constant for reaction with diisopropyl fluorophosphate was reduced 10000-fold, indicating severe alteration in the binding area in W430A. The kcat values for butyrylthiocholine, o-nitrophenyl butyrate, and succinyldithiocholine were lower. This suggested that the mutation had caused misfolding of the active site gorge without altering the Omega loop conformation/dynamics. W430 as well as W231 and W82 appear to form the wall of the active site gorge. Mutation of any of these tryptophans disrupts the architecture of the active site.     

Site-directed mutagenesis of yeast phosphoglycerate kinase. The 'basic-patch' residue arginine 168

There is evidence, some of it of questionable authenticity, which suggests that phosphoglycerate kinase takes up a more compact form following the binding of substrates. Using this evidence it has been assumed that a conformational rearrangement is required for phosphoryl transfer to occur and that this is brought about by moving the enzyme's two domains towards each other. In order to test this hypothesis we have modified, by site-directed mutagenesis, an arginine residue thought to be involved in stabilising the transition-state intermediate. Although some 1.3 nm away from the site of phosphoryl transfer, as seen in the crystallographically determined structure, the substitution of arginine 168 by lysine (R168K) more than halves the specific activity of the enzyme. Substituting the arginine with a methionine (R168M) reduces activity further, but not completely, thus proving that the charge associated with this residue is not essential for catalytic activity. Both mutations raise the Michaelis constants (Km) for ATP and glycerate 3-phosphate. The largest change is observed with the triose substrate and the methionine mutant, suggesting that the primary function of arginine 168 is to influence the environment of this substrate. The effect on activity of adding sulphate to R168K and R168M mutant enzyme has also been investigated. The sulphate activation effect at low substrate concentrations is reduced for the methionine substitution but almost abolished for the lysine substitution. The most reasonable explanation of all these findings is that, in the wild-type enzyme, the guanidinium group of arginine 168 forms a hydrogen bond with one of the triose substrate's C1 oxygens. This steric arrangement would not be possible in the 'open form' of this enzyme as observed in the crystal structure.     

Low-temperature electron transfer from cytochrome to the special pair in Rhodopseudomonas viridis: role of the L162 residue.

Electron transfer from the tetraheme cytochrome c to the special pair of bacteriochlorophylls (P) has been studied by flash absorption spectroscopy in reaction centers isolated from seven strains of the photosynthetic purple bacterium Rhodopseudomonas viridis, where the residue L162, located between the proximal heme c-559 and P, is Y (wild type), F, W, G, M, T, or L. Measurements were performed between 294 K and 8 K, under redox conditions in which the two high-potential hemes of the cytochrome were chemically reduced. At room temperature, the kinetics of P+ reduction include two phases in all of the strains: a dominant very fast phase (VF), and a minor fast phase (F). The VF phase has the following t(1/2): 90 ns (M), 130 ns (W), 135 ns (F), 189 ns (Y; wild type), 200 ns (G), 390 ns (L), and 430 ns (T). These data show that electron transfer is fast whatever the nature of the amino acid at position L162. The amplitudes of both phases decrease suddenly around 200 K in Y, F, and W. The effect of temperature on the extent of fast phases is different in mutants G, M, L, and T, in which electron transfer from c-559 to P+ takes place at cryogenic temperatures in a substantial fraction of the reaction centers (T, 48%; G, 38%; L, 23%, at 40 K; and M, 28%, at 60 K), producing a stable charge separated state. In these nonaromatic mutants the rate of VF electron transfer from cytochrome to P+ is nearly temperature-independent between 294 K and 8 K, remaining very fast at very low temperatures (123 ns at 60 K for M; 251 ns at 40 K for L; 190 ns at 8 K for G, and 458 ns at 8 K for T). In all cases, a decrease in amplitudes of the fast phases is paralleled by an increase in very slow reduction of P+, presumably by back-reaction with Q(A)-. The significance of these results is discussed in relation to electron transfer theories and to freezing at low temperatures of cytochrome structural reorganization.     

Pigment-protein interactions in the photosynthetic reaction centre from Rhodopseudomonas viridis

An X-ray structure analysis of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis provides structural details of the pigment-binding sites. The photosynthetic pigments are found in rather hydrophobic environments provided by the subunits L and M. In addition to apolar interactions, the bacteriochlorophylls of the primary electron donor (`special pair') and the bacteriopheophytins, but not the accessory bacteriochlorophylls, form hydrogen bonds with amino acid side chains of these protein subunits. The two branches of pigments which originate at the primary electron donor, and which mark possible electron pathways across the photosynthetic membrane, are in different environments and show different hydrogen bonding with the protein: this may help to understand why only one branch of pigments is active in the light-driven electron transfer. The primary electron acceptor, a menaquinone (Q_A), is in a pocket formed by the M subunit and interacts with it by hydrophobic contacts and hydrogen bonds. Competitive inhibitors of the secondary quinone Q_B (o-phenanthroline, the herbicide terbutryn) are bound into a pocket provided by the L subunit. Apart from numerous van der Waals interactions they also form hydrogen bonds to the protein.     

Nucleotide Sequences of Genes Coding for Photosynthetic Reaction Centers and Light-Harvesting Proteins of Acidiphilium rubrum and Related Aerobic Acidophilic Bacteria

The nucleotide sequences of the puf operons of the Zn-bacteriochlorophylla(Zn-BChl a)-containing photosynthetic aerobic bacteria, Acidiphiliumrubrum and Acidiphilium angustum, were determined. The nucleotidesequences of the pufL and –M of Acidiphilium cryptum,Acidiphilium multivorum, and Acidiphilium organovorum were alsodetermined. The puf operons of A. rubrum and A. angustum containedpufB, –A,–L, –M, and –C as seen in otherpurple bacteria with an unknown gene directly upstream of pufB.Comparing the deduced amino acid sequences of the puf genesof the Acidiphilium species with those of other purple bacteriashowed that His L168, which is highly conserved in other bacteria,is replaced by a glu-tamic acid in the Acidiphilium species.The three-dimensional structures of the reaction centers ofBlastochloris (Rhodopseudomonas) viridis and Rhodobacter sphaeroidessuggest that this residue locates closely to a special pairof bacteriochlorophylls and may be involved in the stabilizationand function of "Zn-BChl a". The relative content of chargedamino acid residues in the L and M subunit is a little lowerin A. rubrum (10%of total) than in B. viridis (12%), and thetendency is more pronounced in the cyto-chrome subunit: 12.5%in A. rubrum and 18.8% in B. viridis. (Received July 24, 1997; Accepted September 9, 1997)     

Dominant role of local dipolar interactions in phosphate binding to a receptor cleft with an electronegative charge surface: equilibrium, kinetic, and crystallographic studies.

Stringent specificity and complementarity between the receptor, a periplasmic phosphate-binding protein (PBP) with a two-domain structure, and the completely buried and dehydrated phosphate are achieved by hydrogen bonding or dipolar interactions. We recently found that the surface charge potential of the cleft between the two domains that contains the anion binding site is intensely electronegative. This novel finding prompted the study reported here of the effect of ionic strength on the equilibrium and rapid kinetics of phosphate binding. To facilitate this study, Ala197, located on the edge of the cleft, was replaced by a Trp residue (A197W PBP) to generate a fluorescence reporter group. The A197W PBP-phosphate complex retains wild-type Kd and X-ray structure beyond the replacement residue. The Kd (0.18 microM) at no salt is increased by 20-fold at greater than 0.30 M NaCl. Stopped-flow fluorescence kinetic studies indicate a two-step binding process: (1) The phosphate (L) binds, at near diffusion-controlled rate, to the open cleft form (Po) of PBP to produce an intermediate, PoL. This rate decreases with increasing ionic strength. (2) The intermediate isomerizes to the closed-conformation form, PcL. The results indicate that the high specificity, affinity, and rate of phosphate binding are not influenced by the noncomplementary electronegative surface potential of the cleft. That binding depends almost entirely on local dipolar interactions with the receptor has important ramification in electrostatic interactions in protein structures and in ligand recognition.     

Functional Correlation in Amino Acid Residue Mutations of Yeast Iso-2-Cytochrome c that Is Consistent with the Prediction of the Concomitantly Variable Codon Theory in Cytochrome c Evolution

Fitch and Markowitz' theory of concomitantly variable codons (covarions) in evolution predicted the existence of functional correlation in amino acid residue mutations among present-day cytochromes c. Mutational analysis was carried out on yeast iso-2-cytochrome c, where hydrophobic core residues I20, M64, L85, and M98 and surface residue L9 were mutated, in selected combinations, to those found in mammalian and bird cytochromes c. The functionality assay is based upon the ability of yeast cells to grow in YPGE medium. Furthermore, experiments on the single M64L and M98L mutations as well as the double M64L/M98L mutation using NMR showed that the effects of these mutations are to perturb the structural integrity of the protein. We identified functional correlation in two cases of a pair of residue mutations, the I20 V and M98 L pair and the L9 I and L85 I pair. In both cases, only one of the two alternative, putative evolutionary pathways leads to a functional protein and the corresponding pairs of residue mutations are among those found in present-day cytochromes c. Since valine is predicted to be at position 20 in the ancestral form of cytochrome c, the present data provide an explanation for the ancient requirement of leucine rather than methionine in position 98. The present data provide further evidence for the role of those specific atom–atom interactions in directing a pathway in the evolutionary changes of the amino acid sequence that have taken place in cytochrome c, in accordance with Fitch and Markowitz.     

Structure-activity studies of the inhibition of FabI,the enoyl reductase from Escherichia coli,by triclosan: kinetic analysis of mutant FabIs

Triclosan, a common antibacterial additive used in consumer products, is an inhibitor of FabI, the enoyl reductase enzyme from type II bacterial fatty acid biosynthesis. In agreement with previous studies [Ward, W. H., Holdgate, G. A., Rowsell, S., McLean, E. G., Pauptit, R. A., Clayton, E., Nichols, W. W., Colls, J. G., Minshull, C. A., Jude, D. A., Mistry, A., Timms, D., Camble, R., Hales, N. J., Britton, C. J., and Taylor, I. W. (1999) Biochemistry 38, 12514-12525], we report here that triclosan is a slow, reversible, tight binding inhibitor of the FabI from Escherichia coli. Triclosan binds preferentially to the E.NAD(+) form of the wild-type enzyme with a K(1) value of 23 pM. In agreement with genetic selection experiments [McMurry, L. M., Oethinger, M., and Levy, S. B. (1998) Nature 394, 531-532], the affinity of triclosan for the FabI mutants G93V, M159T, and F203L is substantially reduced, binding preferentially to the E.NAD(+) forms of G93V, M159T, and F203L with K(1) values of 0.2 microM, 4 nM, and 0.9 nM, respectively. Triclosan binding to the E.NADH form of F203L can also be detected and is defined by a K(2) value of 51 nM. We have also characterized the Y156F and A197M mutants to compare and contrast the binding of triclosan to InhA, the homologous enoyl reductase from Mycobacterium tuberculosis. As observed for InhA, Y156F FabI has a decreased affinity for triclosan and the inhibitor binds to both E.NAD(+) and E.NADH forms of the enzyme with K(1) and K(2) values of 3 and 30 nM, respectively. The replacement of A197 with Met has no impact on triclosan affinity, indicating that differences in the sequence of the conserved active site loop cannot explain the 10000-fold difference in affinities of FabI and InhA for triclosan.     

Mutagenesis studies of substrate recognition and catalysis in the sortase A transpeptidase from Staphylococcus aureus

The Staphylococcus aureus transpeptidase sortase A (SrtA) is responsible for anchoring a range of virulence- and colonization-associated proteins to the cell wall. SrtA recognizes substrates that contain a C-terminal LPXTG motif. This sequence is cleaved following the threonine, and an amide bond is formed between the threonine and the pentaglycine cross-bridge of branched lipid II. Previous studies have implicated the beta6/beta7 loop region of SrtA in LPXTG recognition but have not systematically characterized this domain. To better understand the individual roles of the residues within this loop, we performed alanine-scanning mutagenesis. Val-168 and Leu-169 were found to be important for substrate recognition, and Glu-171 was also found to be important, consistent with its hypothesized role as a Ca(2+)-binding residue. Gly-167 and Asp-170 were dispensable for catalysis, as was Gln-172. The role of Arg-197 in SrtA has been the subject of much debate. To explore its role in catalysis, we used native chemical ligation to generate semi-synthetic SrtA in which we replaced Arg-197 with citrulline, a non-ionizable analog. This change resulted in a decrease of <3-fold in k(cat)/K(m), indicating that Arg-197 utilizes a hydrogen bond, rather than an electrostatic interaction. Our results are consistent with a model for LPXTG recognition wherein the Leu-Pro sequence is recognized primarily by hydrophobic contacts with SrtA Val-168 and Leu-169, as well as a hydrogen bond from Arg-197. This model contradicts the previously proposed mechanism of binding predicted by the x-ray crystal structure of SrtA.     

Specific mutagenesis of the rieske iron-sulfur protein in Rhodobacter sphaeroides shows that both the thermodynamic gradient and the pK of the oxidized form determine the rate of quinol oxidation by the bc(1) complex

In the Rieske iron-sulfur protein (ISP) of the ubiquinol:cytochrome c(2) oxidoreductase (bc(1) complex) of Rhodobacter sphaeroides, residue Tyr 156 is located close to the iron-sulfur cluster. Previous studies of the equivalent residue in both Saccharomyces cerevisiae [Denke, E., Merbitz-Zahradnik, T., Hatzfeld, O. M., Snyder, C. H., Link, T. A., and Trumpower, B. L. (1998) J. Biol. Chem. 273, 9085-9093] and Paracoccus denitrificans [Schroter, T., Hatzfeld, O. M., Gemeinhardt, S., Korn, M., Friedrich, T., Ludwig, B. , and Link, T. A. (1998) Eur. J. Biochem. 255, 100-106] have indicated that mutations at this site can lead to modifications in the redox potential of the ISP. To study the effect of similar modifications on the thermodynamic behavior and kinetics of partial reactions of the bc(1) complex upon flash activation, we have constructed four mutant strains of Rb. sphaeroides where Tyr 156 was mutated to His, Leu, Phe, or Trp. The bc(1) complex was assembled and able to support photosynthetic growth in all mutants. Three substitutions (Leu, Phe, Trp) led to alteration of the midpoint potential (E(m)) of the ISP and a slowing in rate of quinol oxidation, suggesting that electron transfer from quinol to the oxidized ISP controls the overall rate and that this step includes the high activation barrier. The Trp mutation led to an increase of approximately 1 pH unit in the pK value of the oxidized ISP. The pH dependence of the rate of quinol oxidation in this mutant was also shifted up by approximately 1 pH unit, showing the importance of the protonation state of the ISP for quinol oxidation. This provides support for a model in which the dissociated form of the oxidized ISP is required for formation of the enzyme-substrate complex [Ugulava, N., and Crofts, A. R. (1998) FEBS Lett. 440, 409-413].     

Effect of distance and orientation between arginine-302, histidine-322, and glutamate-325 on the activity of lac permease from Escherichia coli

lac permease of Escherichia coli was modified by site-directed mutagenesis in order to investigate the effects of polarity, distance, and orientation between the components of a putative H+ relay system (Arg302/His322/Glu325) postulated to be involved in lactose-coupled H+ translocation. The importance of polarity between His322 and Glu325 was studied by interchanging the residues, and the modified permease--H322E/E325H--is inactive in all modes of translocation. The effect of distance and/or orientation between His322 and Glu325 was investigated by interchanging Glu325 with Val326, thereby moving the carboxylate one residue around putative helix X. The resulting permease molecule--E325V/V326E--is also completely inactive; control mutations, E325V [Carrasco, N., Püttner, I. B., Antes, L. M., Lee, J. A., Larigan, J. D., Lolkema, J. S., Roepe, P. D., & Kaback, H. R. (1989) Biochemistry (second paper of three in this issue)], and E325A/V326E, indicate that a Glu residue at position 326 inactivates the permease. The wild-type orientation between His and Glu was then restored by further mutation of E325V/V326E to introduce a His residue into position 323 or by interchanging Met323 with His322. The resulting permease molecules--M323H/E325V/V326E and H322M/M323H/E325V/V326E--contain the wild-type His/Glu orientation, but the His/Glu ion pair is rotated about the helical axis by 100 degrees relative to Arg302 in putative helix IX. Both mutants are inactive with respect to all modes of translocation. The results provide strong support for the contention that the polarity between His322 and Glu325 and the geometric relationship between Arg302, His322, and Glu325 are critical for permease activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of mutations of active site residues and amino acids interacting with the Ω loop on substrate activation of butyrylcholinesterase

The peripheral anionic site (PAS) of human butyrylcholinesterase is involved in the mechanism of substrate activation by positively charged substrates and ligands. Two substrate binding loci, D70 in the PAS and W82 in the active site, are connected by the Ω loop. To determine whether the Ω loop plays a role in the signal transduction between the PAS and the active site, residues involved in stabilization of the loop, N83, K339 and W430, were mutated. Mutations N83A and N83Q caused loss of substrate activation, suggesting that N83 which interacts with the D70 backbone may be an element of the transducing system. The K339M and W430A mutant enzymes retained substrate activation. Residues W82, E197, and A328 in the active site gorge have been reported to be involved in substrate activation. At butyrylthiocholine concentrations greater then 2 mM, W82A showed apparent substrate activation. Mutations E197Q and E197G strongly reduced substrate activation, while mutation E197D caused a moderate effect, suggesting that the carboxylate of residue E197 is involved in substrate activation. Mutations A328F and A328Y showed no substrate activation, whereas A328G retained substrate activation. Substrate activation can result from an allosteric effect due to binding of the second substrate molecule on the PAS. Mutation W430A was of special interest because this residue hydrogen bonds to W82 and Y332. W430A had strongly reduced affinity for tetramethylammonium. The bimolecular rate constant for reaction with diisopropyl fluorophosphate was reduced 10 000-fold, indicating severe alteration in the binding area in W430A. The k_cat values for butyrylthiocholine, o-nitrophenyl butyrate, and succinyldithiocholine were lower. This suggested that the mutation had caused misfolding of the active site gorge without altering the Ω loop conformation/dynamics. W430 as well as W231 and W82 appear to form the wall of the active site gorge. Mutation of any of these tryptophans disrupts the architecture of the active site.