Biomedical subjects

G Smulevich

Publications and source records attributed to G Smulevich.

51 records · Page 3Linked to original sources

PubMed1991

Resonance Raman investigation of ferric iron in horseradish peroxidase and its aromatic donor complexes at room and low temperatures.

Resonance Raman (RR) spectra of the acidic form of FeIII horseradish peroxidase (HRP) were obtained at room and low temperatures using B- and Q-band excitation. At 296 K, HRP exhibits two sets of porphyrin skeletal stretching frequencies which are attributed to a thermal mixture of 5- and 6-coordinate high-spin FeIII states. When the temperature is lowered, the observed bands shift to higher frequencies, and these are assigned to intermediate- and low-spin states. Addition of 40% glycerol has no effect on the spectra at 296 K, but at 20 K, all four frequency sets are observed corresponding to the two forms observed at room and low temperature in the absence of glycerol. The 296 K RR spectrum of the HRP-hydroquinone complex is similar to that of free HRP, but conversion to the intermediate- and low-spin states is complete at a higher temperature than in the free enzyme. Addition of benzohydroxamic acid (BHA) to HRP shifts the RR frequencies to those corresponding to a 6-coordinate high-spin species at both room and low temperature. Two upsilon (C = C) stretching modes are observed for HRP and its donor complexes, indicating that the vinyl groups are inequivalent. On BHA binding, one of the vinyl modes and upsilon 37 (Eu) are enhanced, suggesting symmetry lowering of the heme site.

Animals↗

PubMed1990

CO dissociation in cytochrome c peroxidase: site-directed mutagenesis shows that distal Arg 48 influences CO dissociation rates.

To investigate the molecular basis for the 100-fold slower rate of CO dissociation in ferrous peroxidases relative to myoglobin, CO dissociation rates were measured as a function of pH in the cloned cytochrome c peroxidase from yeast [CCP(MI)] and in several mutants in the heme binding pocket prepared by site-directed mutagenesis. The mutants included Asp 235----Asn; Arg 48----Lys, Leu; and His 181----Gly. Changes in the absorption spectrum with pH are consistent with conversion of the CO-ferrous CCP(MI) complex from acidic to alkaline forms by a two-proton cooperative ionization, with an apparent pKa = 7.6, analogous to that described for CCP from bakers' yeast [Iizuka, T., Makino, R., Ishimura, Y., & Yonetani, T. (1985) J. Biol. Chem. 260, 1407-1412]. The rate of CO dissociation (koff) was increased 11-fold (from 0.7 x 10(-4) to 8.0 x 10(-4) s-1) by conversion of the acidic to the alkaline form. Analogous acidic and alkaline forms of the CO complex were also observed in the mutants of CCP(MI) examined here. In the acidic form, koff was increased 5- and 20-fold when Arg 48 was replaced with Lys and Leu, respectively, while in the acidic form of mutants that possess Arg 48, koff was similar to that observed in CCP(MI). Conversion of the CO complex from the acidic to alkaline form increased koff in all the mutants, and the pH-dependent increase in koff correlated with a two-proton cooperative ionization, except in the case of His 181----Gly. In this mutant, pH-dependent increase in koff correlated with a single-proton ionization, implicating His 181 as one of the two residues that is deprotonated in the conversion of CO-ferrous CCP(MI) from acidic to alkaline forms. Only a 2.5-fold variation was observed for koff between the alkaline form of CCP(MI) and the Arg 48----Leu mutant, suggesting that the influence of Arg 48 on the rate of CO dissociation is decreased in the alkaline form by a conformational change.(ABSTRACT TRUNCATED AT 400 WORDS)

Arginine↗

PubMed1990

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)

Cytochrome-c Peroxidase↗

PubMed1990

Probing protein structure and dynamics with resonance Raman spectroscopy: cytochrome c peroxidase and hemoglobin.

Because vibrational frequencies are sensitive to structure, RR spectroscopy can provide structural information about kinetic steps in protein transformations when carried out in a time-resolved mode. UVRR spectroscopy has shown that the aromatic groups of the HbCO photoproduct respond with a delay of 20 microseconds and has provided direct structural evidence that the 20-microseconds kinetic step is the R-T quaternary re-arrangement of the subunits. RR bands of the porphyrin ring show that the core relaxes via a 0.1-microsecond protein motion, which probably allows the Fe atom to attain its full out-of plane displacement. The Fe-His stretching frequency has an elevated value immediately after CO photolysis, in part, perhaps, because of the protein constraint on the Fe displacement. It relaxes on both the 0.1- and 1-microsecond time scales to its value in R-state Hb and then decreases further to its T-state value. These changes may be connected with reorientation of the proximal His side chain. At very early times after a photolysis pulse, heating effects may be an important aspect of the protein dynamics, but further experiments are needed to understand the RR response.

Allosteric Site↗

PubMed1990

Resonance Raman spectroscopy of cytochrome c peroxidase single crystals on a variable-temperature microscope stage.

Good quality resonance Raman (RR) spectra have been obtained for cytochrome c peroxidase single crystals (0.2 x 0.5 x 1 mm) lying on their 110 faces on a microscope stage. Crystal orientation and polarization effects are observed which differentiate the RR bands on the basis of the symmetries of the porphyrin vibrational modes. The measured depolarization ratios are accurately calibrated for isolated bands of both totally symmetric and non totally symmetric modes by using a model of D4h chromophores in an oriented gas using the crystal structure atomic coordinates. The calculations indicate that the electronic transition moments are approximately along the lines connecting the methine bridges, suggesting an electronic steering effect of the vinyl groups. Deviations are observed for bands associated with the porphyrin v10 and the vinyl C = C stretching modes, which may be due to their near-resonant interaction. The band frequencies correspond to those of a five-coordinate high-spin FeIII heme, as previously observed in solution, consistent with the X-ray structure showing the Fe atom to be out of the heme plane on the proximal side with a distal water molecule located at a nonbonded distance, 2.4 A. The temperature dependence of the RR spectrum was determined with a Joule-Thompson cryostat on crystals sealed in glass capillaries. As the temperature is lowered, the spectrum converts to one characteristic of a low-spin FeIII heme. The conversion, which is readily reversible, is quite gradual. It is detectable at -50 degrees C but is incomplete even at -190 degrees C. A temperature effect on the protein structure is proposed which permits the Fe atom to approach the heme plane and bind the distal water molecule, or the distal histidine.

Cytochrome-c Peroxidase↗

PubMed1989

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.

Alkalies↗

PubMed1989

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.

Cytochrome-c Peroxidase↗

PubMed1988

Heme pocket interactions in cytochrome c peroxidase studied by site-directed mutagenesis and resonance Raman spectroscopy.

Resonance Raman spectra are reported for FeII and FeIII forms of cytochrome c peroxidase (CCP) mutants prepared by site-directed mutagenesis and cloning in Escherichia coli. 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. These spectra are used to assess the spin and ligation states of the heme, via the porphyrin marker band frequencies, especially v3, near 1500 cm-1, and, for the FeII forms, the status of the Fe-proximal histidine bond via its stretching frequency. The FeII-His frequency is elevated to approximately 240 cm-1 in CCP(MI) and in all of the distal mutants, due to hydrogen-bonding interactions between the proximal His-175 N delta and the carboxylate acceptor group on Asp-235. The FeII-His RR band has two components, at 233 and 246 cm-1, which are suggested to arise from populations having H-bonded and deprotonated imidazole; these can be viewed in terms of a double-well potential involving proton transfer coupled to protein conformation. The populations shift with changing pH, possibly reflecting structure changes associated with protonation of key histidine residues, and are influenced by the Leu-48 and Phe-191 mutations. A low-spin FeII form is seen at high pH for the Lys-48, Leu-48, Phe-191, and Phe-51 mutants; for the last three species, coordination of the distal His-52 is suggested by a approximately 200-cm-1 RR band assignable to Fe(imidazole)2 stretching.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochrome-c Peroxidase↗

PubMed1988

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)

Arginine↗

PubMed1988

Inclusion complex formation of 1,8-dihydroxyanthraquinone with cyclodextrins in aqueous solution and in solid state.

Complex formation between cyclodextrins and 1,8-dihydroxyanthraquinone in buffer solution has been investigated using absorption, its second derivative (D2), and fluorescence spectroscopy. The results showed that whereas the self-association process was found for 1,8-dihydroxyanthraquinone alone, the monomeric form is microincluded in beta- and gamma-cyclodextrins. The interaction is more favored as the cavity size of cyclodextrins is larger, the molecule being more tightly bound with gamma- than with beta-cyclodextrin. The complex formation inhibits the excited-state intramolecular proton transfer process that has already been reported for 1,8-dihydroxyanthraquinone alone.

Anthraquinones↗

PubMed1986

Transient resonance Raman spectroscopy shows unrelaxed heme following CO photodissociation from cytochrome-c peroxidase.

The 7 ns 436 nm pulses of an H2-shifted YAG laser have been used to photolyze the CO adduct of cytochrome-c peroxidase and produce the resonance Raman spectrum of the photoproduct. A 3 cm-1 downshift, relative to the spectrum of reduced enzyme, was observed for the porphyrin C-N breathing mode, v4. The downshift diminishes with decreasing CO /protein ratio, implying, in conjunction with a recent study of CO binding, that the unrelaxed heme is associated with adduct having a tilted, H-bonded FeCO unit. The downshift is eliminated when the phosphate buffer concentration is increased from 0.01 to 0.1 M. It is proposed that the heme relaxation under study involves a transition between two conformations, B and A, differing in the disposition of the distal residues, and having different v4 frequencies for unligated Fe(II) heme. Conformation B allows H-bonding to bound CO, and is favored at high CO and phosphate concentrations, while conformation A, which is unfavorable to CO H-bonding, is favored at low CO and phosphate concentrations. The recently reported absence of unrelaxed frequencies in the 7 ns photo-product of the CO adduct of horseradish peroxidase has been confirmed, and is attributed to lower stability for conformation B and a smaller A - B v4 difference.

Carbon Monoxide↗

PubMed1986

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.

Carbon Isotopes↗

PubMed1986

Raman and infrared spectra of cytochrome c peroxidase-carbon monoxide adducts in alternative conformational states.

Resonance Raman (RR) spectra are reported for CO-bound cytochrome c peroxidase (CCP). At low pH, two forms are observed: form II, with nu Fe-C = 530 cm-1 and delta FeCO = 585 cm-1, and form I, with nu Fe-C = 495 cm-1 and no detectable delta FeCO. They appear to have coincident nu CO infrared bands, at 1922 cm-1. These low-pH forms, similar to those observed for horseradish peroxidase (HRP), are attributed to tilted, H-bonded CO and perpendicular CO, respectively. The frequencies differ between the two proteins, a weaker H bond to CO being indicated for CCP. As with HRP, the equilibrium between forms I and II is shifted toward the latter at increasing CO concentrations, suggesting that secondary binding of CO perturbs the distal residues. At high pH [8.4, tris(hydroxymethyl)aminomethane buffer] the form II fraction converts to another form, II', with nu FeC = 503 cm-1, delta FeCO = 575 cm-1, and nu CO = 1948 cm-1; a tilted, non-H-bonded geometry is suggested. If phosphate buffer is used, however, form II (H bonded) persists at pH 8.4. This result establishes a role for phosphate in stabilizing the H-bonded form of the enzyme; it is suggested that phosphate binds near the distal imidazole and substantially increases its pKa. The conformational state is also influenced by aging. Fresh protein contains purely high spin FeIII heme, as monitored by the high-frequency RR spectrum, and yields form II almost exclusively at elevated CO concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Monoxide↗

PubMed1985

Nanosecond transient resonance Raman spectra of the FeII-CO and FeIII-NO photolysis products of horseradish peroxidase.

Resonance Raman spectra, obtained with 7 ns pulsed laser excitation, are reported for the photoproducts of the FeII-CO and FeIII-NO adducts of horseradish peroxidase. The porphyrin skeletal frequencies are the same as those observed for unligated FeII and FeIII (native) horseradish peroxidase, respectively. The absence of unrelaxed spectra is discussed in relation to the photoproduct frequency shifts and relaxations observed previously for hemoglobin. It is proposed that protein conformational changes which are likely to be associated with the hydrogen-bonding interactions in the horseradish peroxidase heme pocket may not produce detectable changes in the porphyrin skeletal mode frequencies.

Carbon Monoxide↗

PubMed1980

Raman excitation profiles of actinomycin D.

Pre-resonance Raman spectra of actinomycin D have been measured using the exciting lines of an Ar+ laser. The analysis of the excitation profiles provided information on the origin of the electronic states; in particular, the absorption feature between 400 and 500 nm was interpreted as due to a vibrational structure of a single electronic state which is located at 450 nm. In addition, on the basis of the excitation profiles, the number of observed Raman bands, and their frequencies, it has been possible to propose a vibrational assignment of the chromophoric framework of the drug.

Dactinomycin↗