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PubMed1995

Evidence for a methylammonium-binding site on methylamine dehydrogenase of Thiobacillus versutus.

The nonconvertible substrate analogues di-, tri-, and tetramethylammonium are bound with fairly high affinity to oxidized methylamine dehydrogenase (MADHox) from Thiobacillus versutus and induce the same red-shift in the optical absorbance spectrum of MADHox as do the monovalent cations Cs+, Rb+, and NH4+. Like the monovalent cations, trimethylamine also competitively inhibits the reduction of MADHox by methylamine. Rapid-scan experiments show that within the first few milliseconds of the reaction between MADHox and methylamine a red-shifted intermediate is formed as well. Taken together these experiments demonstrate the existence of a common binding site on MADHox for the substrate CH3NH3+, the substrate analogues (CH3)2NH2+, (CH3)3NH+, and (CH3)4N+, and the monovalent cations Cs+, Rb+, and NH4+. Therefore we conclude that, prior to conversion, methylamine is noncovalently bound to MADHox as a cation. The resonance Raman spectra of MADHox in the absence and presence of Cs+, NH4+, and (CH3)3NH+ are very similar, except for the C=O stretching frequencies of the o-quinone carbonyls of the tryptophyltryptophanquinone (TTQ) active center, which show 5-30 cm-1 downshifts. From these Raman results and the X-ray crystal structure, we conclude that the CH3NH3+ binding site is in close proximity to the O6 carbonyl oxygen of the TTQ.

Binding Sites↗
PubMed1995

Structure and kinetics of formation of catechol complexes of ferric soybean lipoxygenase-1.

Ferric soybean lipoxygenase forms stable complexes with 4-substituted catechols. The structure of the complex between the enzyme and 3,4-dihydroxybenzonitrile has been studied by resonance Raman, electron paramagnetic resonance, visible, and X-ray spectroscopies. It is a bidentate iron-catecholate complex with at least one water ligand. The kinetics of formation of complexes between lipoxygenase and 3,4-dihydroxybenzonitrile and 3,4-dihydroxyacetophenone have been studied by stopped-flow spectroscopy. The data are consistent with two kinetically distinct, reversible steps. The pH dependence of the first step suggests that the substrate for the reaction is the catechol monoanion. When these results are combined, plausible mechanisms for the complexation reaction are suggested.

Catechols↗
PubMed1993

A heme c-peptide model system for the resonance Raman study of c-type cytochromes: characterization of the solvent-dependence of peptide-histidine-heme interactions.

The visible absorption and Soret-excited resonance Raman spectra of ferrous microperoxidase-8 [MP8(II)], an octapeptide containing a heme c, are reported. These spectroscopies indicate that MP8(II), dissolved in aqueous buffered solutions, forms low-spin six-coordinated complexes in the 7-14 pH range. Intermolecular bonding interactions of MP8(II) in water account for this behavior. On the contrary, when the hemopeptide is dispersed in aqueous solutions containing detergent or an alcohol, the spectroscopic data show that the iron atom of MP8(II) is essentially high-spin five-coordinated in accordance with a monomeric structure of MP8(II). In addition to a high-spin signature to the heme skeletal modes, the high-frequency regions of resonance Raman spectra characterize an electronic influence of the thioether bridges on the frequency of stretching modes of C beta-C beta bonds (nu 2, nu 11, and nu 29). On the other hand, the low-frequency Raman spectra of monomeric MP8(II) at pH 7.5 present significant differences in the 150-250-cm-1 regions depending upon the solvent composition (pH, presence or absence of detergent, alcohol). These effects are attributed to frequency variations of the Fe-N(His)-involving mode which indicate changes in the H-bonding interactions of the axial His and therefore solvent-dependent changes of the octapeptide conformation. Our resonance Raman data further show that the axial His of monomeric MP8(II) could be totally deprotonated in aqueous cetyltrimethylammonium bromide solution at very alkaline pH (pKa = 13.3). The vibrational data (100-1700 cm-1) obtained for the various monomeric forms of MP8(II) are expected to be useful for determining the heme structure and environment in reduced c'-type cytochromes. Comparisons of resonance Raman data with X-ray crystallographic data available for different hemoproteins allow us to evaluate the ionization and H-bonding states of the His bound to the high-spin five-coordinated hemes. These data are discussed in terms of proximal influence of protein-His-heme interactions on the determination and the regulation of a particular biological function.

Amino Acid Sequence↗
PubMed1993

Quantitative structural comparisons of heme protein crystals and solutions using resonance Raman spectroscopy.

Resonance Raman difference spectra have been used to compare crystal and solution samples of metmyoglobin (metMb), deoxymyoglobin (deoxyMb), and cytochrome P450. At pH 6.0, the frequency shifts of the heme core size sensitive bands v2, v3, and v4 were determined to be less than 0.3, 1.0, and 0.3 cm-1, respectively, for metMb and to be less than 1.0, 1.0, and 0.3 cm-1, respectively, for deoxyMb. This shows that the heme core size differences between the crystal and solution conformations are less than 0.002 A for metMb and less than 0.003 A for deoxyMb. These results disagree with a recent extended X-ray absorption fine structure study [Zhang, K., Chance, B., Reddy, K. S., Ayene, I., Stern, E. A., & Bunker, G. (1991) Biochemistry 30, 9116-9120] which claims that a 0.05-A difference exists in the average iron-ligand distance between the crystalline and solution forms of metMb at pH 6.5. At pH 8.5, metMb solution samples change gradually from a predominantly high-spin to a predominantly low-spin species as the ammonium sulfate concentration is increased to the level found in the crystal mother liquor. No Raman frequency shifts are found between the crystal and solution forms of metMb at pH 8.5 when the ammonium sulfate concentrations are equal. On the other hand, for deoxyMb, we find a significant alteration in the 220/240-cm-1 line shape and relative intensities, suggesting that some histidine-heme perturbation takes place upon crystallization.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonium Sulfate↗
PubMed1991

Characterization of the tryptophan-derived quinone cofactor of methylamine dehydrogenase by resonance Raman spectroscopy.

The resonance Raman (RR) spectrum of oxidized methylamine dehydrogenase (MADHOX) exhibits a set of C-H, C-C, C = C, and C = O vibrational modes between 900 and 1700 cm-1 that are characteristic of the quinone moiety of the tryptophan tryptophlyquinone (TTQ) cofactor. The close similarity of the RR spectra for MADHs from Paracoccus denitrificans (Pd), Thiobacillus versutus (Tv), and bacterium W3A1 proves that the same cofactor is present in all three proteins. The MADHs from Pd and Tv have a v(C = O) mode at approximately 1625 cm-1 that shifts approximately 20 cm-1 upon 18O substitution of one of the carbonyl oxygens and is assigned to the in-phase symmetric stretch of the two C = O groups. The semiquinone form of Pd MADH has its own characteristic RR spectrum with altered peak frequencies and intensities as well as a decrease in the total number of peaks. The hydroxide and ammonia adducts of MADHOX produce RR spectra similar to that of the semiquinone. The spectral changes in all three cases are interpreted as being due to reduced conjugation of the cofactor. The ammonia adduct is formulated as a carbinolamine, a likely intermediate in the enzymatic mechanism. In contrast, formation of the electron-transfer complex between amicyanin and MADHOX has no effect on the vibrational frequencies (and, hence, structure) of either the MADH quinone or the amicyanin blue copper site. The behavior of the TTQ cofactors of Pd and Tv MADHs are very similar to one another and somewhat different from W3A1 MADH, particularly with regard to adduct formation and ability to undergo isotope exchange with solvent. These differences are ascribed to the cofactor environments within the proteins rather than to the structure of the cofactor itself.

Ammonia↗
PubMed1985

Iron(III)-adriamycin and Iron(III)-daunorubicin complexes: physicochemical characteristics, interaction with DNA, and antitumor activity.

Fe(III) complexes of two anthracyclines, adriamycin and daunorubicin, have been studied. Using potentiometric and spectroscopic measurements, we have shown that adriamycin and daunorubicin form two well-defined species with Fe(III), which can be formulated as respectively Fe(HAd)3 and Fe(HDr)3. In these formulas, HAd and HDr stand for adriamycin and daunorubicin in which the 1,4-dihydroxy-anthraquinone moiety is half-deprotonated. Both complexes are six-membered chelates. The stability constant is beta = (2.5 +/- 0.5) X 10(28) for both complexes. Interaction with DNA has been studied showing that, despite strong coordination to Fe(III), anthracyclines are able to intercalate between DNA bases pairs, releasing the metal. These complexes display antitumor activity against P 388 leukemia that compares with that of the free drug. Fe(HAd)3, unlike adriamycin, does not catalyze the flow of electrons from NADH to molecular oxygen through NADH dehydrogenase. Moreover, it is shown that the triferric adriamycin compound so called "quelamycin" is in fact a mixture of Fe(HAd)3 and polymeric ferric hydroxide.

Circular Dichroism↗
PubMed1988

Identification and properties of an oxoferryl structure in myeloperoxidase compound II.

Myeloperoxidase compound II has been characterized by using optical absorption and resonance Raman spectroscopies. Compared to compounds II in other peroxidases, the electronic and vibrational properties of this intermediate are strongly perturbed due to the unusual active-site iron chromophore that occurs in myeloperoxidase. Despite this difference in prosthetic group, however, other properties of myeloperoxidase compound II are similar to those observed for this intermediate in the more common peroxidases (horseradish peroxidase in particular). Two forms of the myeloperoxidase intermediate species, each with distinct absorption spectra, are recognized as a function of pH. We present evidence consistent with interconversion of these two forms via a heme-linked ionization of a distal amino acid residue with a pKa congruent to 9. From resonance Raman studies of isotopically labeled species at pH 10.7, we identify an iron-oxygen stretching frequency at 782 cm-1, indicating the presence of an oxoferryl (O = FeIV) group in myeloperoxidase compound II. We further conclude that the oxo ligand is not hydrogen bonded above the pKa but possibly exhibits oxygen exchange with the medium at pH values below the pKa due to hydrogen bonding of the oxo ligand to the distal protein group.

Humans↗
PubMed2004

Reactions of spinach nitrite reductase with its substrate, nitrite, and a putative intermediate, hydroxylamine.

Plant nitrite reductase (NiR) catalyzes the reduction of nitrite (NO(2)(-)) to ammonia, using reduced ferredoxin as the electron donor. NiR contains a [4Fe-4S] cluster and an Fe-siroheme, which is the nitrite binding site. In the enzyme's as-isolated form ([4Fe-4S](2+)/Fe(3+)), resonance Raman spectroscopy indicated that the siroheme is in the high-spin ferric hexacoordinated state with a weak sixth axial ligand. Kinetic and spectroscopic experiments showed that the reaction of NiR with NO(2)(-) results in an unexpectedly EPR-silent complex formed in a single step with a rate constant of 0.45 +/- 0.01 s(-)(1). This binding rate is slow compared to that expected from the NiR turnover rates reported in the literature, suggesting that binding of NO(2)(-) to the as-isolated form of NiR is not the predominant type of substrate binding during enzyme turnover. Resonance Raman spectroscopic characterization of this complex indicated that (i) the siroheme iron is low-spin hexacoordinated ferric, (ii) the ligand coordination is unusually heterogeneous, and (iii) the ligand is not nitric oxide, most likely NO(2)(-). The reaction of oxidized NiR with hydroxylamine (NH(2)OH), a putative intermediate, results in a ferrous siroheme-NO complex that is spectroscopically identical to the one observed during NiR turnover. Resonance Raman and absorption spectroscopy data show that the reaction of oxidized NiR ([4Fe-4S](2+)/Fe(3+)) with hydroxylamine is binding-limited, while the NH(2)OH conversion to nitric oxide is much faster.

Hydroxylamine↗
PubMed2004

Cytochrome rC552, formed during expression of the truncated, Thermus thermophilus cytochrome c552 gene in the cytoplasm of Escherichia coli, reacts spontaneously to form protein-bound 2-formyl-4-vinyl (Spirographis) heme.

Expression of the truncated (lacking an N-terminal signal sequence) structural gene of Thermus thermophilus cytochrome c(552) in the cytoplasm of Escherichia coli yields both dimeric (rC(557)) and monomeric (rC(552)) cytochrome c-like proteins [Keightley, J. A., et al. (1998) J. Biol. Chem. 273, 12006-12016], which form spontaneously without the involvement of cytochrome c maturation factors. Cytochrome rC(557) is comprised of a dimer and has been structurally characterized [McRee, D., et al. (2001) J. Biol. Chem. 276, 6537-6544]. Unexpectedly, the monomeric rC(552) transforms spontaneously to a cytochrome-like chromophore having, in its reduced state, the Q(oo) transition (alpha-band) at 572 nm (therefore called p572). The X-ray crystallographic structure of rC(552), at 1.41 A resolution, shows that the 2-vinyl group of heme ring I is converted to a [heme-CO-CH(2)-S-CH(2)-C(alpha)] conjugate with cysteine 11. Electron density maps obtained from isomorphous crystals of p572 at 1.61 A resolution reveal that the 2-vinyl group has been oxidized to a formyl group. This explains the lower energy of the Q(oo)() transition, the presence of a new, high-frequency band in the resonance Raman spectra at 1666 cm(-1) for oxidized and at 1646 cm(-1) for reduced samples, and the greatly altered, paramagnetically shifted (1)H NMR spectrum observed for this species. The overall process defines a novel mechanism for oxidation of the 2-vinyl group to a 2-formyl group and adds to the surprising array of chemical reactions that occur in the interaction of heme with the CXXCH sequence motif in apocytochromes c.

Circular Dichroism↗
PubMed2005

The heme of cystathionine beta-synthase likely undergoes a thermally induced redox-mediated ligand switch.

Cystathionine beta-synthase (CBS) is a pyridoxal-5'-dependent enzyme that catalyzes the condensation of homocysteine and serine to form cystathionine. Human CBS is unique in that heme is also required for maximal activity, although the function of heme in this enzyme is presently unclear. The study presented herein reveals that the heme of human CBS undergoes a coordination change upon reduction at elevated temperatures. We have termed this new species "CBS424" and demonstrate that its formation is likely irreversible when pH 9 Fe(III) CBS is reduced at moderately elevated temperatures (approximately 40 degrees C and higher) or when pH 9 Fe(II) CBS is heated to similar temperatures. Spectroscopic techniques, including resonance Raman, electronic absorption, and variable temperature/variable field magnetic circular dichroism spectroscopy, provide strong evidence that CBS424 is coordinated by two neutral donor ligands. It appears likely that the native cysteine(thiolate) heme ligand is displaced by an endogenous neutral donor upon conversion to CBS424. This behavior is consistent with other six-coordinate, cysteine(thiolate)-ligated heme centers, which seek to avoid this coordination structure in the Fe(II) state. Functional assays show that CBS424 is inactive and suggest that the ligand switch is responsible for eliminating enzyme activity. When this investigation is taken together with other functional studies of CBS, it provides strong evidence that coordination of Cys52 to the heme iron is crucial for full activity in this enzyme. We hypothesize that cysteine displacement may serve as a mechanism for CBS inactivation and that second-sphere interactions of the Cys52 thiolate with surrounding residues are responsible for communicating the heme ligand displacement to the CBS active site.

Circular Dichroism↗
PubMed1997

Threonine-89 participates in the active site of bacteriorhodopsin: evidence for a role in color regulation and Schiff base proton transfer.

Bacteriorhodopsin (bR) functions as a light-driven proton pump in the purple membrane of Halobacterium salinarium. A major feature of bR is the existence of an active site which includes a retinylidene Schiff base and amino acid residues Asp-85, Asp-212, and Arg-82. This active site participates in proton transfers and regulates the visible absorption of bacteriorhodopsin and its photointermediates. In this work we find evidence that Thr-89 also participates in this active site. The substitution Thr-89 --> Asn (T89N) results in changes in the properties of the all-trans retinylidene chromophore of light-adapted bR including a redshift of the visible lambda(max) and a downshift in C=N and C=C stretch frequencies. Changes are also found in the M and N intermediates of the T89N photocycle including shifts in lambda(max), a downshift of the Asp-85 carboxylic acid C=O stretch frequency by 10 cm(-1), and a 3-5-fold decrease in the rate of formation of the M intermediate. In contrast, the properties of the 13-cis retinylidene chromophore of dark-adapted T89N as well as the K and L intermediates of the T89N photocycle are similar to the wild-type bacteriorhodopsin. These results are consistent with an interaction of the hydroxyl group of Thr-89 with the protonated Schiff base of light-adapted bR and possibly the N intermediate but not the 13-cis chromophore of dark-adapted bR or the K and L intermediates. Thr-89 also appears to influence the rate of Schiff base proton transfer to Asp-85 during formation of the M intermediate, possibly through an interaction with Asp-85. In contrast, the hydroxyl group of Thr-89 is not obligatory for proton transfer from Asp-96 to the Schiff base during formation of the N intermediate.

Bacteriorhodopsins↗
PubMed1998

Generation of triplet and cation-radical bacteriochlorophyll a in carotenoidless LH1 and LH2 antenna complexes from Rhodobacter sphaeroides.

The LH1 antenna complex and a native form of the LH2 complex were isolated from the carotenoidless R26 and R26.1 mutants of Rhodobacter sphaeroides by the use of a new detergent, sucrose monocholate. One-color, pump-and-probe transient Raman spectroscopy of these complexes using 351 nm, approximately 50 ps pulses showed the generation of the triplet state of bacteriochlorophyll a (BChl a), whereas measurements using 355 nm, approximately 12 ns pulses showed the generation of BChl a cation radical. Subpicosecond to nanosecond time-resolved absorption spectroscopy using 388 nm, 200 fs pulses for excitation showed rapid (<1 ps) generation of the triplet state and fast decay (<10 ps) of the singlet state of BChl a. Microsecond absorption spectroscopy confirmed the generation of BChl a cation radical. EPR spectroscopy using 532 nm, approximately 5 ns pulses for excitation established the generation of BChl a cation radical. The EPR line width suggested that the unpaired electron is shared by two BChl a molecules. In LH1, the yield of BChl a cation radical per complex was estimated to be about 80% of that in the reaction center, and in LH2 about 50%. Thus, rapid generation of the triplet state, and its subsequent transformation into the cation-radical state of BChl a have been shown to be intrinsic properties of B870 and B850 BChl a assembly in the carotenoidless LH1 and LH2 antenna complexes. In the case of the carotenoid-containing LH2 complex, the triplet states of BChl a and carotenoid (spheroidene) were generated immediately after excitation, but the triplet-state BChl a was quenched efficiently by the carotenoid so that no BChl a cation radical was generated. Thus, the photoprotective function of the carotenoid in this antenna complex is shown.

Bacterial Proteins↗
PubMed2005

X-ray absorption spectroscopy measurements of liquid water.

Recent studies, based on X-ray absorption spectroscopy (XAS) and X-ray Raman scattering (XRS), have shown that the hydrogen bond network in liquid water consists mainly of water molecules with only two strong hydrogen bonds. Since this result is controversial, it is important to demonstrate the reliability of the experimental data, which is the purpose of this paper. Here we compare X-ray absorption spectra of liquid water recorded with five very different techniques sensitive to the local environment of the absorbing molecule. Overall, the spectra obtained with photon detection show a very close similarity and even the observable minor differences can be understood. The comparison demonstrates that XAS and XRS can indeed be applied reliably to study the local bonding of the water molecule and thus to reveal the hydrogen bond situation in bulk water.

Absorptiometry, Photon↗
PubMed2006

Fe vibrational spectroscopy of myoglobin and cytochrome f.

The Fe vibrational density of states (VDOS) has been determined for the heme proteins deoxymyoglobin, metmyoglobin, and cytochrome f in the oxidized and reduced states, using nuclear resonance vibrational spectroscopy (NRVS). For cytochrome f in particular, the NRVS spectrum is compared with multiwavelength resonance Raman spectra to identify those Raman modes with significant Fe displacement. Modes not seen by Raman due to optical selection rules appear in the NRVS spectrum. The mean Fe force constant extracted from the VDOS illustrates how Fe dynamics varies among these four monoheme proteins, and is correlated with oxidation and spin state trends seen in model heme compounds. The protein's contribution to Fe motion is dominant at low frequencies, where coupling to the backbone tightly constrains Fe displacements in cytochrome f, in contrast to enhanced heme flexibility in myoglobin.

Cytochromes f↗
PubMed2006

Irreducible representation and projection operator application to understanding nonlinear optical phenomena: hyper-Raman, sum frequency generation, and four-wave mixing spectroscopy.

Symmetry plays an essential role in understanding optical activities of a molecule in infrared and Raman vibrational spectroscopy as well as in nonlinear optical vibrational spectroscopy. Each vibrational mode belongs to an irreducible representation of the underlying symmetry group. In this paper, using the alpha-helical polypeptide symmetry as an example, we calculate all the third rank nonzero hyper-Raman tensors as well as the infrared and Raman tensors by applying the projection operators to each irreducible species. We demonstrate that the projection operator method provides selection rules for the infrared, Raman, and hyper-Raman vibrational transitions and also other nonlinear optical spectroscopy such as sum frequency generation and the four-, five-, and six-wave mixing coherent vibrational transitions. Specific expressions for all nonzero elements of the corresponding nonlinear susceptibility tensors in a laboratory-fixed coordinate frame are also deduced.

Optics and Photonics↗
PubMed2006

Angle-dependent terahertz time-domain spectroscopy of amino acid single crystals.

The measurement of absorption spectra using angle-dependent terahertz (THz) time-domain spectroscopy for amino acid single crystals of l-cysteine and l-histidine is reported for the first time. Linearly polarized THz radiation enables us to observe angle-dependent far-infrared absorption spectra of amino acid single crystals and determine the direction of the oscillating dipole of the molecules in the 20-100 cm(-1) range. By comparing the THz spectra of a single crystal and powder, we found that there was a clear hydrogen-bond peak in the crystal spectrum as a result of the larger hydrogen-bond network. The low-temperature THz spectra of amino acid microcrystals showed more intermolecular vibrational modes than those measured at room temperature. An ab initio frequency calculation of a single amino acid molecule was used to predict the intramolecular vibrational modes. The validity of the calculation models was confirmed by comparing the results with experimentally obtained data in the Raman spectral region.

Amino Acids↗
PubMed2006

Nitrogen 1s near-edge X-ray absorption fine structure spectroscopy of amino acids: Resolving zwitterionic effects.

Considerable variation is observed in the near-edge X-ray absorption fine structure (NEXAFS) spectra of amino acids. To unambiguously characterize the chemical origin of this variation, we have acquired the nitrogen 1s NEXAFS spectra of several amino acids and other model compounds and complemented these experimental measurements with ab initio calculations of isolated molecules and molecular clusters. The systematic differences observed between the zwitterionic and un-ionized forms of amino acids arise directly from the structural difference (-NH2 vs -NH3+), which leads to a change in the degree of Rydberg-valence mixing. Further change arises from quenching of this Rydberg character in the spectra of condensed amino acids. Ab initio calculations are used to explore the degree of Rydberg-valence mixing in the solid state.

Algorithms↗