Cytochrome c peroxidase mutant active site structures probed by resonance Raman and infrared signatures of the CO adducts

Vibrational frequencies associated with FeC and CO stretching and FeCO bending modes have been determined via resonance Raman (RR) and infrared (IR) spectroscopy for cytochrome c peroxidase (CCP) mutants prepared by site-directed mutagenesis. These include the bacterial "wild type", CCP(MI), and mutations involving groups on the proximal (Asp-235----Asn; Trp-191---Phe) and distal (Trp-51----Phe; Arg-48----Leu and Lys) side of the heme. The data were analyzed with the aid of a recently established correlation between nu FeC and nu CO, which can be used to distinguish between back-bonding and axial ligand donor effects. At high pH all adducts showed essentially the same vibrational pattern (form I') with nu FeC approximately 505 cm-1, nu CO approximately 1948 cm-1, and delta FeCO (weak RR band) approximately 576 cm-1. These frequencies are very similar to those shown by the myoglobin CO adduct and imply a "normal" H-bond of the proximal histidine. At pH 7 (pH 6 for Asn-235 and Leu-48), different forms are seen for different proteins: form I (nu FeC approximately 500 cm-1, nu CO = 1922-1941 cm-1, and delta FeCO approximately 580 cm-1, very weak) in the case of CCP(MI) and Phe-191, as well as bakers' yeast CCP, or form II (nu FeC approximately 530 cm-1, nu CO = 1922-1933 cm-1, and delta FeCO = 585 cm-1, moderately strong) for Asn-235 and Phe-51.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational change and histidine control of heme chemistry in cytochrome c peroxidase: resonance Raman evidence from Leu-52 and Gly-181 mutants of cytochrome c peroxidase.

Resonance Raman (RR) spectra are reported for Fe(III), Fe(II), and Fe(II)CO forms of site-directed mutants of the cytochrome c peroxidase variant CCP(MI), cloned in Escherichia coli. The Fe(II) form is five-coordinate (5-c) and high-spin at low pH, but it is six-coordinate (6-c) and low-spin at high pH except when the distal His-52 residue is replaced with Leu, showing the sixth ligand to be the His-52 imidazole. Although the Leu-52 mutant stays 5-c, it does undergo an alkaline transition, as revealed by upshifts and broadening of bands assigned to vinyl C = C stretching (1620 cm-1) and C beta-vinyl bending (402 cm-1). Similar changes are seen for CCP(MI) and other mutants. Thus the alkaline transition induces a conformational change that affects the vinyl groups, probably through changes in their orientation, and that permits the His-52 imidazole to bind the Fe. The RR band arising from the stretching of the proximal Fe(II)-imidazole bond contains components at ca. 235 and 245 cm-1 for CCP(MI), which are believed to reflect a double well potential for the H-bond between the proximal His-175 imidazole and the Asp-235 carboxylate group. Loss of this H-bond by mutation of Asp-235 to Asn results in the loss of these two bands and their replacement by a single band at 205 cm-1. Although the Fe(II)-imidazole stretching mode cannot be observed in the 6-c alkaline form of the enzyme, the sixth ligand in the alkaline form of CCP(MI) is photolabile, and the status of the Fe(II)-imidazole bond can be determined in the resulting 5-c-photoproduct. For CCP(MI) at alkaline pH, the conformation change induces an increase in the 235/245-cm-1 ratio, reflecting a perturbation of the H-bond potential. In the His-52----Leu mutant, a 205-cm-1 band appears along with the 235/245-cm-1 doublet at alkaline pH, indicating partial loss of the proximal H-bond due to the distal alteration. The effect of mutations that perturb the H-bonding network that extends from the distal to the proximal side of the heme is more dramatic: at alkaline pH, His-181----Gly, Arg-48----Leu, and Trp-51----Phe mutants show an Fe(II)-imidazole stretching mode at 205 cm-1 exclusively, indicating complete loss of the proximal Asp-235-His-175 H-bond.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of temperature and glycerol on the resonance Raman spectra of cytochrome c peroxidase and selected mutants

The high-frequency resonance Raman spectra of FeIII yeast native cytochrome c peroxidase (CCP) and five of its mutants [CCP(MI), Phe-51, Leu-48, Lys-48, Asn-235, and Phe-191] were recorded in phosphate buffer, pH 7.0, and in glycerol/phosphate mixtures at 295 and 10 K. Glycerol induces heme coordination changes in some of the CCP mutants at room temperature. It apparently weakens the binding of the Fe atom to ligands in the distal heme cavity and drives the heme toward the 5-coordinate, high-spin state. At 10 K, native CCP and all the mutants (except Phe-51 which remains 6-coordinate, high-spin) show various distributions of spin and coordination states which differ from those observed at 295 K. Upon cooling in phosphate buffer, pH 7, and to a much lesser extent in 66% glycerol/phosphate, an internal strong-field ligand is coordinated to the Fe. A likely candidate is H2O-595, which could become a strong-field ligand on H-bonding and/or proton transfer to H2O-648, and/or the distal His-52. However, distal His-52 itself cannot be ruled out as the coordinating ligand considering that the Phe-51 mutant, which binds H2O-595 at room temperature, does not show a large 6-coordinate, low-spin component at 10 K like the other mutants. These results clearly indicate that the Fe coordination in CCP and its mutants is sensitive to both temperature and solvent composition.     

X-ray structures of recombinant yeast cytochrome c peroxidase and three heme-cleft mutants prepared by site-directed mutagenesis

The 2.2-A X-ray structure for CCP(MI), a plasmid-encoded form of Saccharomyces cerevisiae cytochrome c peroxidase (CCP) expressed in Escherichia coli [Fishel, L.A., Villafranca, J. E., Mauro, J. M., & Kraut, J. (1987) Biochemistry 26, 351-360], has been solved, together with the structures of three specifically designed single-site heme-cleft mutants. The structure of CCP(MI) was solved by using molecular replacement methods, since its crystals grow differently from the crystals of CCP isolated from bakers' yeast used previously for structural solution. Small distal-side differences between CCP(MI) and bakers' yeast CCP are observed, presumably due to a strain-specific Thr-53----Ile substitution in CCP(MI). A Trp-51----Phe mutant remains pentacoordinated and exhibits only minor distal structural adjustments. The observation of a vacant sixth coordination site in this structure differs from the results of solution resonance Raman studies, which predict hexacoordinated high-spin iron [Smulevich, G., Mauro, J.M., Fishel, L. A., English, A. M., Kraut, J., & Spiro, T. G. (1988) Biochemistry 27, 5477-5485]. The coordination behavior of this W51F mutant is apparently altered in the presence of a precipitating agent, 30% 2-methyl-2,4-pentanediol. A proximal Trp-191----Phe mutant that has substantially diminished enzyme activity and altered magnetic properties [Mauro, J. M., Fishel, L. F., Hazzard, J. T., Meyer, T. E., Tollin, G., Cusanovich, M. A., & Kraut, J. (1988) Biochemistry 27, 6243-6256] accommodates the substitution by allowing the side chain of Phe-191, together with the segment of backbone to which it is attached, to move toward the heme. This relatively large (ca. 1 A) local perturbation is accompanied by numerous small adjustments resulting in a slight overall compression of the enzyme's proximal domain; however, the iron coordination sphere is essentially unchanged. This structure rules out a major alteration in protein conformation as a reason for the dramatically decreased activity of the W191F mutant. Changing proximal Asp-235 to Asn results in two significant localized structural changes. First, the heme iron moves toward the porphyrin plane, and distal water 595 now clearly resides in the iron coordination sphere at a distance of 2.0 A. The observation of hexacoordinated iron for the D235N mutant is in accord with previous resonance Raman results. Second, the indole side chain of Trp-191 has flipped over as a result of the mutation; the tryptophan N epsilon takes part in a new hydrogen bond with the backbone carbonyl oxygen of Leu-177.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparative proton NMR analysis of wild-type cytochrome c peroxidase from yeast, the recombinant enzyme from Escherichia coli, and an Asp-235----Asn-235 mutant

Proton NMR spectra of cytochrome c peroxidase (CcP) isolated from yeast (wild type) and two Escherichia coli expressed proteins, the parent expressed protein [CcP(MI)] and the site-directed mutant CcP(MI,D235N) (Asp-235----Asn-235), have been examined. At neutral pH and in the presence of only potassium phosphate buffer and potassium nitrate, wild-type Ccp and CcP(MI) demonstrate nearly identical spectra corresponding to normal (i.e., "unaged") high-spin ferric peroxidase. In contrast, the mutant protein displays a spectrum characteristic of a low-spin form, probably a result of hydroxide ligation. Asp-235 is hydrogen-bonded to the proximal heme ligand, His-175. Changing Asp-235 to Asn results in alteration of the pK for formation of the basic form of CcP. Thus, changes in proximal side structure mediate the chemistry of the distal ligand binding site. All three proteins bind F-, N3-, and CN- ions, although the affinity of the mutant protein (D235N) for fluoride ion appears to be much higher than that of the other two proteins. Analysis of proton NMR spectra of the cyanide ligated forms leads to the conclusion that the mutant protein (D235N) possesses a more neutral proximal histidine imidazole ring than does either wild-type CcP or CcP(MI). It confirms that an important feature of the cytochrome c peroxidase structure is at least partial, and probably full, imidazolate character for the proximal histidine (His-175).     

Replacement of the axial histidine ligand with imidazole in cytochrome c peroxidase. 1. Effects on structure

Replacement of the axial histidine ligand with exogenous imidazole has been accomplished in a number of heme protein mutants, where it often serves to complement the functional properties of the protein. In this paper, we describe the effects of pH and buffer ion on the crystal structure of the H175G mutant of cytochrome c peroxidase, in which the histidine tether between the heme and the protein backbone is replaced by bound imidazole. The structures show that imidazole can occupy the proximal H175G cavity under a number of experimental conditions, but that the details of the interaction with the protein and the coordination to the heme are markedly dependent on conditions. Replacement of the tethered histidine ligand with imidazole permits the heme to shift slightly in its pocket, allowing it to adopt either a planar or distally domed conformation. H175G crystallized from both high phosphate and imidazole concentrations exists as a novel, 5-coordinate phosphate bound state, in which the proximal imidazole is dissociated and the distal phosphate is coordinated to the iron. To accommodate this bound phosphate, the side chains of His-52 and Asn-82 alter their positions and a significant conformational change in the surrounding protein backbone occurs. In the absence of phosphate, imidazole binds to the proximal H175G cavity in a pH-dependent fashion. At pH 7, imidazole is directly coordinated to the heme (d(Fe--Im) = 2.0 A) with a nearby distal water (d(Fe--HOH) = 2.4 A). This is similar to the structure of WT CCP except that the iron lies closer in the heme plane, and the hydrogen bond between imidazole and Asp-235 (d(Im--Asp) = 3.1 A) is longer than for WT CCP (d(His--Asp) = 2.9 A). As the pH is dropped to 5, imidazole dissociates from the heme (d(Fe--Im) = 2.9 A), but remains in the proximal cavity where it is strongly hydrogen bonded to Asp-235 (d(Im--Asp) = 2.8 A). In addition, the heme is significantly domed toward the distal pocket where it may coordinate a water molecule. Finally, the structure of H175G/Im, pH 6, at low temperature (100 K) is very similar to that at room temperature, except that the water above the distal heme face is not present. This study concludes that steric restrictions imposed by the covalently tethered histidine restrain the heme and its ligand coordination from distortions that would arise in the absence of the restricted tether. Coupled with the functional and spectroscopic properties described in the following paper in this issue, these structures help to illustrate how the delicate and critical interactions between protein, ligand, and metal modulate the function of heme enzymes.     

Photodissociable endogenous ligand in alkaline-reduced cytochrome c peroxidase implicates distal protein tension

Laser excitation of alkaline- (pH 8.5) reduced cytochrome c peroxidase (CCP) produces resonance Raman (RR) bands arising from both low- and high-spin heme species (nu 3 = 1493/1471 cm-1) even though in the absence of laser excitation the absorption spectrum is characteristic of a purely low-spin species. The high-spin fraction is higher in a stationary than in a rotating sample, indicating that the high-spin contribution arises from photolysis induced by the Raman laser. This conclusion was confirmed by monitoring the absorption spectrum during laser irradiation. Photolability of the low-spin form is somewhat less than that of the CO adduct. The endogenous photolabile ligand is proposed to be the distal histidine residue, His-52. Recent picosecond absorption measurements (Jongeward et al., 1988) show that imidazole ligands in heme proteins do photodissociate but recombine in picoseconds, leading to net photostability on longer time scales. It is proposed that a fraction of the His-52 residues recombine much more slowly in CCP because of protein strain in the ligated form. This strain can also explain the anomalously rapid rate of CO binding to alkaline CCP.     

Single-crystal resonance Raman spectroscopy of site-directed mutants of cytochrome c peroxidase

Resonance Raman spectra are reported for single crystals of cytochrome c peroxidase (CCP) mutants, taken by using a microscope equipped with a variable-temperature stage. The spectra are similar to those observed for the mutant proteins in solution, but there are detectable differences having to do with the coordination and spin state of the heme. The Asn-235 mutant contains a mixture of six-coordinate high- and low-spin states with a detectably higher fraction of the former than in solution. Upon cooling even to 223 K, the heme is converted mostly to the low-spin form. The Phe-191 mutant likewise shows a high/low-spin six-coordinate mixture, together with a preponderant population of five-coordinate heme. Upon cooling, the high-spin six-coordinate population converts immediately to the low-spin form, while the five-coordinate population does so more slowly. This behavior is intermediate between that of native CCP and the Asn-235 mutant, consistent with an ancillary role for the normal Trp-191-Asp-235 H-bond in the proximal anchoring of the heme Fe. The Phe-51 mutant shows a dominant high-spin five-coordinate heme population in the single crystal, whereas in solution the six-coordinate form is dominant. This difference is mimicked by adding 2-methyl-2,4-pentanediol (MPD) to the solution and is attributed to the dehydrating effect of MPD, which is present during crystallization. Upon lowering the temperature, the five-coordinate heme converts partially to a six-coordinate high-spin form.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resonance Raman evidence that distal histidine protonation removes the steric hindrance to upright binding of carbon monoxide by myoglobin

The resonance Raman band assigned to Fe--CO stretching in the sperm whale myoglobin CO adduct shifts from 507 cm-1 at neutral pH to 488 cm-1 at low pH, in concert with a shift of the C-O stretching infrared band from 1947 to 1967 cm-1 (Fuchsman & Appleby, 1979), while the 575-cm-1 Fe-C-O bending RR band loses intensity. The pKa that characterizes these changes is approximately 4.4. The vibrational frequencies at low pH are well modeled by the protein-free CO, imidazole adduct of protoheme in a nonpolar solvent while those at high pH are modeled by the adduct of a heme with a covalent strap (Yu et al., 1983) which inhibits upright CO binding. It is inferred that the Fe-C-O unit changes from a tilted to an upright geometry when the distal histidine is protonated, because its side chain swings out of the heme pocket due to electrostatic repulsion with a nearby arginine residue. A different protonation step (pKa = 5.7), which has been shown to modulate the CO rebinding kinetics (Doster et al., 1982) as well as the optical spectrum (Fuchsman & Appleby, 1979), is suggested to involve a global structure change associated with protonation of histidine residues distant from the heme.     

Alternative carbon monoxide binding modes for horseradish peroxidase studied by resonance Raman spectroscopy

Resonance Raman (RR) spectroscopy and infrared spectroscopy have been used to characterize the three vibrational modes, CO and FeC stretching and FeCO bending, for carbon monoxide bound to reduced horseradish peroxidase, with the aid of 13CO and C18O isotope shifts. At high pH, one species, I, is observed, with nu FeC = 490 cm-1 and nu CO = 1932 cm-1. The absence of a band attributable to delta FeCO suggests a linear FeCO unit normal to the heme plane. The data were consistent with I having a strongly H-bonded proximal histidine, as shown by a comparison with imidazole and imidazolate adducts of FeIIPPDME(CO) (PPDME = protoporphyrin IX dimethyl ester), with nu FeC = 497 and 492 cm-1 and nu CO = 1960 and 1942 cm-1. At low pH an additional species, II, is observed, with nu FeC = 537 cm-1, nu CO = 1904 cm-1, and delta FeCO = 587 cm-1; it is attributed to FeCO that is H bonded to a protonated distal histidine, the H bond strongly lowering nu CO and raising nu FeC. The appearance of delta FeCO in the RR spectrum suggests that the FeCO unit in II is tilted with respect to the heme plane. At low pH, the population of I and II depends on the CO concentration. I dominates at low CO/protein levels but is replaced by II as the amount of CO is increased. This behavior is suggested to arise from secondary binding of CO, which induces a conformation change involving the distal residues of the heme pocket.     

Crystal structure of nitric oxide inhibited cytochrome c peroxidase

We have collected X-ray diffraction data from a crystal of cytochrome c peroxidase (CCP) complexed with the inhibitor nitric oxide to a resolution of 2.55 A. A difference Fourier map shows density indicating the NO ligand is bound to the heme iron at the sixth coordination site in a bent configuration. Structural adjustments were determined by least-squares refinement that yielded an agreement residual of R = 0.18. The orientation of the ligand, tilting toward Arg-48, causes adjustment in the position of this nearby polar side chain. As a model for the substrate hydrogen peroxide, this geometry is consistent with the suggestion that Arg-48 serves to polarize the O-O peroxide bond to promote heterolytic cleavage of the bond [Poulos, T. L., & Kraut, J. (1980) J. Biol. Chem. 255, 8199-8205]. Strong difference density is also observed near residues 190-194, especially around the indole ring of Trp-191. The density indicates movement of the indole ring away from the proximal His-175 imidazole ring by about 0.25 A, which appears to cause perturbation of the neighboring residues. The response of Trp-191 on the proximal side of the heme to binding nitric oxide on the distal side probably results from delocalization of the electron density of the ligand. Relevant to this is the recent finding that a mutant in which Trp-191 is replaced by phenylalanine has dramatically reduced activity, less than 0.05% of the parent activity [Mauro, J. M., Fishel, L. A., Hazzard, J. T., Meyer, T. E., Tollin, G., Cusanovich, M. A., & Kraut, J. (1988) Biochemistry 27, 6243-6256].(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton NMR assignments of heme contacts and catalytically implicated amino acids in cyanide-ligated cytochrome c peroxidase determined from one- and two-dimensional nuclear Overhauser effects.

Proton NMR assignments of the heme pocket and catalytically relevant amino acid protons have been accomplished for cyanide-ligated yeast cytochrome c peroxidase. This form of the protein, while not enzymatically active itself, is the best model available (that displays a resolvable proton NMR spectrum) for the six-coordinate low-spin active intermediates, compounds I and II. The assignments were made with a combination of one- and two-dimensional nuclear Overhauser effect methods and demonstrate the utility of NOESY experiments for paramagnetic proteins of relatively large size (Mr 34,000). Assignments of both isotope exchangeable and nonexchangeable proton resonances were obtained by using enzyme preparations in both 90% H2O/10% D2O and, separately, in 99.9% D2O solvent systems. Complete resonance assignments have been achieved for the proximal histidine, His-175, and His-52, which is a member of the catalytic triad on the distal side of the heme. In addition, partial assignments are reported for Trp-51 and Arg-48, catalytically important residues, both on the distal side. Aside from His-175, partial assignments for amino acids on the proximal side of the heme are proposed for the alanines at primary sequence positions 174 and 176 and for Thr-180 and Leu-232.     

Crystal structure of yeast cytochrome c peroxidase refined at 1.7-A resolution

The crystal structure of cytochrome c peroxidase (EC 1.11.1.5) has been refined to an R factor of 0.20 computed for all reflections to 1.7 A. The refined molecular model includes 263 bound water molecules and allows for x-ray scattering by amorphous solvent. The mean positional error in atomic coordinates is estimated to lie between 0.12 and 0.21 A. Two factors are identified which may account for the ability of the enzyme to stabilize high-oxidation states of the heme iron during catalysis: 1) the proximal histidine forms a hydrogen bond with a buried aspartic acid side chain, Asp-235; and 2) the heme environment is more polar than in the cytochromes c or globins, owing to the presence of the partially buried side-chain of Arg-48 and five water molecules bound in close proximity to the heme. Two of these occupy the presumed peroxide-binding site. Two candidates are likely for the side chain that is oxidized to a free radical during formation of Compound I: 1) Trp-51, which rests 3.3 A above the heme plane in close proximity (2.7 A) to the sixth coordination position; and 2) Met-172, which is 3.7 A from the heme. Nucleophilic stabilization of the methionyl cation radical may be possible via Asp-235. His-181 is found to lie coplanar with the heme in a niche between the two propionates near the suspected cytochrome c-binding site. A network of hydrogen bonds involving this histidine may provide a preferred pathway for electron transfer between hemes.     

Ligand binding and structural perturbations in cytochrome c peroxidase. A crystallographic study

Crystal structures of the complexes formed between cytochrome c peroxidase and cyanide, nitric oxide, carbon monoxide, and fluoride have been determined and refined to 1.85 A. In all four complexes significant changes occur in the distal heme pocket due to movement of Arg-48, His-52, and a rearrangement of active site water molecules. In the cyanide, nitric oxide, and carbon monoxide complexes, Arg-48 moves away from the ligand while in the fluoride complex Arg-48 moves in toward the ligand to form a hydrogen bond or ion pair with the fluoride. More subtle changes occur on the proximal side of the heme. In an earlier study at lower resolution (Edwards, S. L., Kraut, J., and Poulos, T. L. (1988) Biochemistry 27, 8074-8081), we found that nitric oxide binding causes perturbations in the proximal domain involving Trp-191 which has been confirmed by the present study. Trp-191 is stacked parallel to and in contact with the proximal ligand, His-175. Nitric oxide binding results in a slight movement of Trp-191 away from His-175 and a large increase in crystallographic temperature factors indicating increased mobility of these residues on the proximal side of the heme. These proximal-side changes are unique to nitric oxide and are not related strictly to spin-state or oxidation state of the iron atom since similar changes were not observed in the cyanide (low-spin ferric), carbon monoxide (low-spin ferrous), or fluoride (high-spin ferric) complexes.     

Heme coordination states of unfolded ferrous cytochrome C

The structural changes of ferrous Cyt-c that are induced by binding to SDS micelles, phospholipid vesicles, DeTAB, and GuHCl as well as by high temperatures and changes in the pH have been studied by RR and UV-Vis absorption spectroscopies. Four species have been identified in which the native methionine-80 ligand is removed from the heme iron. This coordination site is either occupied by a histidine (His-33 or His-26) to form a 6cLS configuration, which is the prevailing species in GuHCl at pH 7.0 and ambient temperature, or remains vacant to yield a 5cHS configuration. The three identified 5cHS species differ with respect to the hydrogen-bond interactions of the proximal histidine ligand (His-18) and include a nonhydrogen-bonded, a hydrogen-bonded, and a deprotonated imidazole ring. These structural motifs have been found irrespective of the unfolding conditions used. An unambiguous spectroscopic distinction of these 5cHS species is possible on the basis of the Fe-N(imidazole) stretching vibrations, the RR bands in the region between 1300 and 1650 cm(-1), and the electronic transitions in the Soret- and Q-band regions. In acid and neutral solutions, the species with a hydrogen-bonded and a nonhydrogen-bonded His-18 prevail, whereas in alkaline solutions a configuration with a deprotonated His-18 ligand is also observed. Upon lowering the pH or increasing the temperature in GuHCl solutions, the structure on the proximal side of the heme is perturbed, resulting in a loss of the hydrogen-bond interactions of the His-18 ligand. Conversely, the hydrogen-bonded His-18 of ferrous Cyt-c is stabilized by electrostatic interactions which increase in strength from phospholipid vesicles to SDS micelles. The results here suggest that unfolding of Cyt-c is initiated by the rupture of the Fe-Met-80 bond and structural reorganizations on the distal side of the heme pocket, whereas the proximal part is only affected in a later stage of the denaturation process.     

Lactose carrier mutants of Escherichia coli with changes in sugar recognition (lactose versus melibiose).

The purpose of this research was to identify amino acid residues that mediate substrate recognition in the lactose carrier of Escherichia coli. The lactose carrier transports the alpha-galactoside sugar melibiose as well as the beta-galactoside sugar lactose. Mutants from cells containing the lac genes on an F factor were selected by the ability to grow on succinate in the presence of the toxic galactoside beta-thio-o-nitrophenylgalactoside. Mutants that grew on melibiose minimal plates but failed to grow on lactose minimal plates were picked. In sugar transport assays, mutant cells showed the striking result of having low levels of lactose downhill transport but high levels of melibiose downhill transport. Accumulation (uphill) of melibiose was completely defective in all of the mutants. Kinetic analysis of melibiose transport in the mutants showed either no change or a greater than normal apparent affinity for melibiose. PCR was used to amplify the lacY DNA of each mutant, which was then sequenced by the Sanger method. The following six mutations were found in the lacY structural genes of individual mutants: Tyr-26-->Asp, Phe-27-->Tyr, Phe-29-->Leu, Asp-240-->Val, Leu-321-->Gln, and His-322-->Tyr. We conclude from these experiments that Tyr-26, Phe-27, Phe-29 (helix 1), Asp-240 (helix 7), Leu-321, and His-322 (helix 10) either directly or indirectly mediate sugar recognition in the lactose carrier of E. coli.     

Essential histidines of prostaglandin endoperoxide synthase. His-309 is involved in heme binding

Prostaglandin endoperoxide (PGH) synthase has a single iron protoporphyrin IX which is required for both the cyclooxygenase and peroxidase activities of the enzyme. At room temperature, the heme iron is coordinated at the axial position by an imidazole, and about 20% of the heme iron is coordinated at the distal position by an imidazole. We have used site-directed mutagenesis to investigate which histidine residues are involved in PGH synthase catalysis and heme binding. Individual mutant cDNAs for ovine PGH synthases were prepared with amino acid substitutions at each of 13 conserved histidines. cos-1 cells were transfected with each of these cDNAs, and the cyclooxygenase and peroxidase activities of the resulting microsomal PGH synthases were measured. Mutant PGH synthases in which His-207, His-309, or His-388 was replaced with either glutamine or alanine lacked both activities. Gln-386 and Ala-386 PGH synthase mutants exhibited cyclooxygenase but not peroxidase activities. Other mutants exhibited both activities at varying levels. Because binding of heme renders native PGh synthase resistant to cleavage by trypsin, we examined the effects of heme on the relative sensitivities of native, Ala-204, Ala-207, Ala-309, Ala-386, and Ala-388 mutant PGH synthases to trypsin as a measure of the heme-protein interaction. The Ala-309 PGh synthase mutant was notably hypersensitive to tryptic cleavage, even in the presence of exogenous heme; in contrast, the native enzyme and the other alanine mutants exhibited similar, lower sensitivities toward trypsin and, except for the Ala-386 mutant, were partially protected from trypsin cleavage by heme. Preincubation of the native and each of the alanine mutant PGH synthases, including the Ala-309 mutant, with indomethacin protected the proteins from trypsin cleavage. Thus, all the mutant proteins retain sufficient three-dimensional structure to bind cyclooxygenase inhibitors. Our results suggest that His-309 is one of the heme ligands, probably the axial ligand, of PGH synthase. Two other histidines, His-207 and His-388, are essential for both PGH synthase activities suggesting that either His-207 or His-388 can serve as the distal heme ligand; however, the trypsin cleavage measurements imply that neither His-207 nor His-388 is required for heme binding. This is consistent with the fact that only 20% of the distal coordination position of the heme iron of PGH synthase is occupied by an imidazole side chain.     

Isolation of four novel tachykinins from frog (Rana catesbeiana) brain and intestine

In a survey for unknown bioactive peptides in frog (Rana catesbeiana) brain and intestine, we isolated four novel peptides that exhibit potent stimulant effects on smooth muscle preparation of guinea pig ileum. By microsequencing and synthesis, these peptides were identified as Lys- Pro- Ser- Pro- Asp- Arg- Phe- Tyr- Gly- Leu- Met- NH2 (ranatachykinin A), Tyr- Lys- Ser- Asp- Ser- Phe- Tyr- Gly- Leu- Met- NH2 (ranatachykinin B), His- Asn- Pro- Ala- Ser- Phe- Ile- Gly- Leu- Met- NH2 (ranatachykinin C) and Lys- Pro- Ans- Pro- Glu- Arg- Phe- Tyr- Ala- Pro- Met- NH2 (ranatachykinin D). Ranatachykinin (RTK) A, B and C conserve the C- terminal sequence, Phe- X- Gly- Leu- Met- NH2, which is common to known members of the tachykinin family. On the other hand, RTK-D has a striking feature in its C-terminal sequence, Phe- Tyr- Ala- Pro- Met- NH2, which has never been found in other known tachykinins, and may constitute a new subclass in the tachykinin family.     

Conformation of cobrotoxin in aqueous solution as studied by nuclear magnetic resonance.

The 270-MHz proton NMR spectra of cobrotoxin from Naja naja atra were observed in 2H2O solution. The pKa value (5.93) of His-32 is slightly lower than the pKa value (6.65) of the reference model of N-acetylhistidine methylamide, because of the electrostatic interaction with Arg-33 and Asp-31. The pKa value (5.3--5.4) of His-4 is appreciably low, because of the interaction with the positively charged guanidino group possibly of Arg-59. The hydrogen-deuterium exchange rates in 2H2O solution were measured of cobrotoxin and imidazole-bearing models. The second-order rate constants of N-acetylhistidine methylamide, N-acetylhistidine and imidazole acetic acid satisfy the Br?nsted relation. With reference to this Br?nsted relation, the imidazole ring of His-32 is confirmed to be exposed. The imidazole ring of His-4 is also exposed and the exchange rate is excessively promoted by the presence possibly of Arg-59 in the proximity. All the methyl proton resonances are assigned to amino-acid types, by conventional double-resonance method and more effectively by the spin-echo double-resonance method. Eight methyl proton resonances are identified as due to the gamma and/or delta-methyl groups of Val-46, Leu-1, Ile-50 and Ile-52 residues. The proximity of aromatic ring protons and methyl protons is elucidated by the analyses of nulcear Overhauser effect enhancements. The aromatic proton resonances of Trp-29 are affected by the ionizable groups of Asp-31, His-32 and Tyr-35. The methyl groups of Ile-50 are in the proximity to the aromatic ring of Trp-29 and the methyl groups of Ile-52 are in the proximity to Tyr-25. The highest-field methyl proton resonance is due to a threonine residue in the proximity to His-4. The appreciable temperature-dependent chemical shift of this methyl proton resonance suggests a temperature-dependent local conformational equilibrium around the His-4 residue of the first loop of the cobrotoxin molecule.