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T G Spiro

Publications and source records attributed to T G Spiro.

At least 55 records · Page 3Linked to original sources
PubMed1994

Nature's carbonylation catalyst: Raman spectroscopic evidence that carbon monoxide binds to iron, not nickel, in CO dehydrogenase.

Carbon monoxide dehydrogenase catalyzes the synthesis of acetyl-coenzyme A from coenzyme A, a methyl group, and carbon monoxide. The carbon monoxide binds to a mixed metal center of the enzyme, which contains nickel bridged to an iron-sulfur cluster. Resonance Raman spectroscopy has been used to identify both C-O stretching and metal-CO stretching vibrations of the carbon monoxide adduct of the enzyme. This adduct was shown by isotopic exchange to be on the pathway for acetyl-coenzyme A synthesis. The metal to which carbon monoxide is bound was established to be iron, not nickel, by preparation of enzyme from bacteria grown on iron-54 and nickel-64. The Fe-CO frequency is low, 360 wave numbers, implying a weak bond, probably because of electron donation from sulfide and thiolate ligands of the iron. A bimetallic mechanism is proposed, in which carbon monoxide binds to an iron atom and is subsequently attacked by a methyl group on a nearby nickel atom, forming an acetyl ligand, which is then transferred to coenzyme A.

Acetyl Coenzyme A↗
PubMed1993

R and T states of fluoromethemoglobin probed by ultraviolet resonance Raman spectroscopy.

Ultraviolet resonance Raman spectra are reported for the fluoride complex of methemoglobin (metHbF), with and without added inositol hexaphosphate (IHP), an allosteric effector known to stabilize the T quaternary structure. Environmental changes in Trp and Tyr residues give rise to the difference features, which are similar to those observed in the difference spectrum between deoxyHb and the CO adduct, consistent with T-state formation in metHbF in the presence of IHP. There are, however, important differences. The intensities of the difference signals are attenuated by about one-third, indicating a lower T-state population in the IHP-bound metHbF than in native deoxyHb. And a new signal is seen which arises from the interior tryptophans, probably reflecting a change in their H-bond status associated with the presence of fluoride as the sixth heme ligand in the T state. Implications of these results for the nature of the molecular forces opposing ligation in the T state are discussed.

Hemoglobins↗
PubMed1992

Substrate polarization by residues in delta 5-3-ketosteroid isomerase probed by site-directed mutagenesis and UV resonance Raman spectroscopy.

delta 5-3-Ketosteroid isomerase (KSI: EC 5.3.3.1) of Pseudomonas testosteroni catalyzes the isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by the stereospecific transfer of the steroid 4 beta-proton to the 6 beta-position, using Tyr-14 as a general acid and Asp-38 as a base. Ultraviolet resonance Raman (UVRR) spectra have been obtained for the catalytically active double mutant Y55F + Y88F, which retains Tyr-14 as the only tyrosine residue (referred to as the Y14(0) mutant), and the Y14F mutant, which has 50,000-fold lower activity. The UVRR results establish that binding of the product analog and competitive inhibitors 19-nortestosterone or 4-fluoro-19-nortestosterone to the Y14(0) mutant does not result in the formation of deprotonated Tyr-14. The UVRR spectra of the steroid inhibitors show large decreases in the vinyl and carbonyl stretching frequencies on binding to the Y14(0) enzyme but not on binding to the Y14F enzyme. These changes cannot be mimicked by protonation of the steroids. For 19-nortestosterone, the vinyl and carbonyl stretching frequencies shift down (with respect to the values in aqueous solution) by 18 and 27 cm-1, respectively, on binding to Y14(0) KSI. It is proposed that the changes in the steroid resonance Raman spectrum arise from polarization of the enone moiety via the close proximity of the charged Asp-38 side chain to the vinyl group and the directional hydrogen bond between Tyr-14 and the 3-carbonyl oxygen of the steroid enone. The 230-nm-excited UVRR spectra do not, however, show changes that are characteristic of strong hydrogen bonding from the tyrosine hydrogen. It is proposed that this hydrogen bonding is compensated by a second hydrogen bond to the Tyr-14 oxygen from another protein residue. UVRR spectra of the Y14(0) enzyme obtained using 200 nm excitation show enhancement of the amide II and S Raman bands. The secondary structure of KSI was estimated from the amide II and S intensities and was found to be low in alpha-helical structure. The alpha-helix content was estimated to be in the range of 0-25% (i.e., 10 +/- 15%).

Binding, Competitive↗
PubMed1991

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)

Alkenes↗
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 evidence for low-spin Fe2+ heme a3 in energized cytochrome c oxidase: implications for the inhibition of O2 reduction.

Resonance Raman (RR) spectra are reported for reduced submitochondrial particles (SMP) with excitation at 441.6 nm, where Raman bands of the cytochrome c oxidase heme a groups are selectively enhanced. Addition of ATP to energize the membranes induces the formation of a new band at 1644 cm-1 and partial loss of intensity in a band at 1567 cm-1. These changes are modeled by adding cyanide to reduced cytochrome c oxidase and are attributed to partial conversion of cytochrome (cyt) a3 from a high-spin to a low-spin state. This conversion is abolished by addition of excess oligomycin, an ATPase inhibitor, or FCCP, an uncoupler of proton translocation, and is reversed when the ATP is consumed. The observed spin-state conversion is attributed to the binding of an endogenous ligand to the cyt a3 Fe atom. This ligation is suggested to be induced by a local increase in pH and/or by a global conformation change associated with the generation of a transmembrane potential. Since O2 binding requires a vacant coordination site at cyt a3, the ligation of this site must retard O2 reduction and could thus provide a simple mechanism for energy-linked regulation of respiration. No changes in the RR spectrum were observed upon adding Ca2+ or H+ to reduced cytochrome c oxidase. The cyt a3 spin-state change associated with membrane energization is unrelated to the cyt a absorption red shift induced by adding Ca2+ or H+ to cytochrome c oxidase.

Animals↗
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

Resonance Raman studies of Escherichia coli sulfite reductase hemoprotein. 1. Siroheme vibrational modes.

Resonance Raman (RR) spectra are reported for the hemoprotein subunit (SiR-HP) of Escherichia coli NADPH-sulfite reductase (EC 1.8.1.2) in various ligation and redox states. Comparison of the RR spectra of extracted siroheme and the mu-oxo FeIII dimer of octaethylisobacteriochlorin with those of mu-oxo FeIII octaethylchlorin dimer and mu-oxo FeIII octaethylporphyrin dimer demonstrates that many siroheme bands can be correlated with established porphyrin skeletal modes. Depolarization measurements are a powerful tool in this correlation, since the 45 degrees rotation of the C2 symmetry axis of the isobacteriochlorin ring relative to the chlorin system results in reversal of the polarization properties (polarized vs anomalously polarized) of bands correlating with B1g and B2g modes of porphyrin. Various SiR-HP adducts (CO, NO, CN-, SO3(2-] show upshifted high-frequency bands, characteristic of the low-spin state and consistent with the expected core size sensitivity of the skeletal modes. Fully reduced unliganded SiR-HP (both siroheme and Fe4S4 cluster reduced) in liquid solution displays RR features comparable to those of high-spin ferrous porphyrins; on freezing, the RR spectrum changes, reflecting an apparent mixture of siroheme spin states. At intermediate reduction levels in solution a RR species is observed whose high-frequency bands are upshifted relative to oxidized and fully reduced SiR-HP. This spectrum, thought to arise from the "one-electron" state of SiR-HP (siroheme reduced, cluster oxidized), may be due to S = 1 FeII siroheme.

Binding Sites↗
PubMed1989

Resonance Raman studies of Escherichia coli sulfite reductase hemoprotein. 2. Fe4S4 cluster vibrational modes.

Resonance Raman (RR) spectra from the hemoprotein subunit of Escherichia coli sulfite reductase (SiR-HP) are examined in the low-frequency (200-500 cm-1) region where Fe-S stretching modes are expected. In spectra obtained with excitation in the siroheme Soret or Q bands, this region is dominated by siroheme modes. Modes assignable to the Fe4S4 cluster are selectively enhanced, however, with excitation at 488.0 or 457.9 nm. The assignments are confirmed by observation of the expected frequency shifts in SiR-HP extracted from E. coli grown on 34S-labeled sulfate. The mode frequencies and isotopic shifts resemble those seen in RR spectra of other Fe4S4 proteins and analogues, but the breathing mode of the cluster at 342 cm-1 is higher than that observed in the other species. Spectra of various ligand complexes of SiR-HP reveal only slight sensitivity of the cluster terminal ligand modes to the presence of exogenous heme ligands, at variance with a model of ligand binding in a bridged mode between heme and cluster. Close examination of RR spectra obtained with siroheme Soret-band excitation reveals additional 34S-sensitive features at 352 and 393 cm-1. These may be attributed to a bridging thiolate ligand.

Escherichia coli↗
PubMed1989

Resonance Raman studies of Escherichia coli sulfite reductase hemoprotein. 3. Bound ligand vibrational modes.

The vibrations of the bound diatomic heme ligands CO, CN-, and NO are investigated by resonance Raman spectroscopy in various redox states of Escherichia coli sulfite reductase hemoprotein, and assignments are generated by use of isotopically labeled ligands. For the fully reduced CO complex (ferrous siroheme, reduced Fe4S4 cluster) at room temperature, nu CO is observed at 1904 cm-1, shifting to 1920 cm-1 upon oxidation of the cluster. The corresponding delta FeCO modes are identified at 574 and 566 cm-1, respectively, by virtue of the zigzag pattern of their isotopic shifts. In frozen solution, two species are observed for the cluster-oxidized state, with nu CO at 1910 and 1936 cm-1 and nu FeC at 532 and 504 cm-1, respectively; nu FeC for the fully reduced species is identified at 526 cm-1 in the frozen state. For the ferrous siroheme-NO complex (cluster oxidized), nu NO is identified at 1555 cm-1 in frozen solution and a low-frequency mode is identified at 558 cm-1; this stretching mode is significantly lower than that observed in Mb-NO. For the ferric siroheme cyanide complexes evidence of two ligand-bonding forms is observed, with modes at 451/390 and 451/352 cm-1; they are distinguished by a reversal of the isotopic shift patterns of the upper and lower modes and could arise from a linear and a bent Fe-C unit, respectively. For the ferrous siroheme cyanide complex isotope-sensitive modes observed at 495 and 452 cm-1 are assigned to the FeCN- bending and FeC stretching vibrations, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Isotopes↗
PubMed1989

Ionic strength dependence of cytochrome c structure and Trp-59 H/D exchange from ultraviolet resonance Raman spectroscopy.

Ultraviolet resonance Raman spectra are reported for cytochrome c (cyt c) in FeII and FeIII oxidation states at low (0.005 M) and high (0.9-1.5 M) ionic strength. With 200-nm excitation the amide band intensities are shown to remain constant, establishing that redox state and ionic strength have no influence on the alpha-helical content. The tyrosine 830/850-cm-1 doublet, however, shows a loss in 830-cm-1 intensity at I = 0.005 M for the FeIII protein, suggesting a weakening or a loss of H-bonding from an internal tyrosine, probably Tyr-48, which is H-bonded to a heme propionate group in cyt c crystals. Excitation profiles of tryptophan peak at approximately 229 nm for both FeII and FeIII forms of cyt c, but at approximately 218 nm for aqueous tryptophan. The approximately 2200-cm-1 red shift of the resonant electronic transition is attributed to the Trp-59 residue being buried and H-bonded. Consistent with this Trp environment, the H-bond-sensitive 877-cm-1 Trp band is strong and sharp, and the 1357/1341-cm-1 doublet has a large intensity ratio, approximately 1.5, for both FeII and FeIII cyt c. The 877-cm-1-band frequency shifts to 860 cm-1 when the Trp indole proton is replaced by a deuteron. This band was used to show that Trp H/D exchange in D2O is much faster for FeIII than FeII cyt c. The half-time for exchange at room temperature is estimated to be approximately 30 and approximately 5 h, respectively, for FeII and FeIII when examined at I = 0.005.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Ultraviolet resonance Raman spectroscopy of distamycin complexes with poly(dA)-poly(dT) and poly(dA-dT): role of H-bonding.

Raman spectra are reported for distamycin, excited at 320 nm, in resonance with the first strong absorption band of the chromophore. Qualitative band assignments to pyrrole ring and amide modes are made on the basis of frequency shifts observed in D2O. When distamycin is dissolved in dimethyl sulfoxide or dimethylformamide, large (30 cm-1) upshifts are seen for the band assigned to amide I, while amides II and III shift down appreciably. Similar but smaller shifts are seen when distamycin is bound to poly(dA-dT) and poly(dA)-poly(dT). Examination of literature data for N-methylacetamide in various solvents shows that the amide I frequencies correlate well with solvent acceptor number but poorly with solvent donor number. This behavior implies that acceptor interactions with the C = O group are more important than donor interactions with the N-H group in polarizing the amide bond and stabilizing the zwitterionic resonance form. The resonance Raman spectra therefore imply that the distamycin C = O groups, despite being exposed to solvent, are less strongly H-bonded in the polynucleotide complexes than in aqueous distamycin, perhaps because of orienting influences of the nearby backbone phosphate groups. In this respect, the poly(dA-dT) and poly(dA)-poly(dT) complexes are the same, showing the same RR frequencies. Resonance Raman spectra were also obtained at 200-nm excitation, where modes of the DNA residues are enhanced. The spectra were essentially the same with and without distamycin, except for a perceptable narrowing of the adenine modes of poly(dA-dT), suggesting a reduction in conformational flexibility of the polymer upon drug binding.

Binding Sites↗
PubMed1989

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.

Allosteric Regulation↗
PubMed1988

Tyrosine hydrogen-bonding and environmental effects in proteins probed by ultraviolet resonance Raman spectroscopy.

Ultraviolet resonance Raman spectra with 229-nm excitation are reported for aqueous tyrosine and for ovomucoid third domain proteins from chicken [OMCHI3(-)] and from chachalaca [OMCHA(-)], as well as alpha 1-, alpha 2-, and beta-purothionin. At this excitation wavelength interference from phenylalanine is minimized, and it is possible to determine the frequencies of the Tyr ring modes nu 8a and nu 8b. The nu 8b frequency decreases with the degree of Tyr H-bond donation, reaching a limiting value for deprotonated tyrosine. This spectroscopic indicator of H-bond strength was calibrated by using the model compound p-cresol in H-bond acceptor solutions for which the enthalpy of H-bond formation can be obtained from the literature. With this calibration it is possible to estimate Tyr H-bond enthalpies in proteins for which Tyr is a H-bond donor; values of 13.7, 9.6, and 11.2 kcal/mol were found for OMCHA3(-) and for alpha 1- (or alpha 2-) and beta-purothionin, respectively. The intensity of the 1176-cm-1 nu 9a band of Tyr excited at 229 nm and also the intensity ratio of the Tyr 830/850-cm-1 Fermi doublet excited at 200 nm both correlate strongly with the estimated H-bond enthalpies, but large deviations are seen for the purothionins, reflecting a special environment for the Tyr residue of these proteins, which is believed to be constrained in a hydrophobic pocket. The molar intensity of the strong approximately 1000-cm-1 nu 12 band of phenylalanine in aqueous solution is about half the value observed in most proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗