Biomedical subjects

J Sanders-Loehr

Publications and source records attributed to J Sanders-Loehr.

54 records · Page 3Linked to original sources

PubMed1990

Activation of the iron-containing B2 protein of ribonucleotide reductase by hydrogen peroxide.

The active form of protein B2, the small subunit of ribonucleotide reductase, contains two dinuclear Fe(III) centers and a tyrosyl radical. The inactive metB2 form also contains the same diferric complexes but lacks the tyrosyl radical. We now demonstrate that incubation of metB2 with hydrogen peroxide generates the tyrosyl radical. The reaction is optimal at 5.5 nM hydrogen peroxide, with a maximum of 25-30% tyrosyl radical being formed after approximately 1.5 hr of incubation. The activation reaction is counteracted by a hydrogen peroxide-dependent reduction of the tyrosyl radical. It is likely that the generation of the radical proceeds via a ferryl intermediate, as in the proposed mechanisms for cytochrome P-450 and the peroxidases.

Enzyme Activation↗

PubMed1989

Resonance Raman spectroscopy of ribonucleotide reductase. Evidence for a deprotonated tyrosyl radical and photochemistry of the binuclear iron center.

Native ribonucleotide reductase from Escherichia coli exhibits a resonance-enhanced Raman mode at 1498 cm-1 that is characteristic of a tyrosyl radical. The Raman frequency as well as the absorption maximum at 410 nm identifies the radical as being in a deprotonated state. The B2 subunit of ribonucleotide reductase shows an additional resonance Raman mode at 493 cm-1 that has been assigned to the symmetric stretch of an Fe-O-Fe moiety. When samples of active B2 or metB2 are exposed to a tightly focused laser beam at 406.7 nm, there is a loss of intensity at 493 cm-1 and the appearance of a new peak at 595 cm-1. Although the 595-cm-1 feature was previously assigned to an Fe-OH vibration on the basis of its 23-cm-1 shift to lower energy in H2(18)O and the apparent dependence of its intensity on pH [Sjöberg, B. M., Loehr, T. M., & Sanders-Loehr, J. (1987) Biochemistry 26, 4242], the present studies indicate that the intensity of this mode is dependent primarily on input laser power. The peak at 595 cm-1 is more plausibly assigned to a new vs(Fe-O-Fe) mode in view of its lack of the deuterium isotope dependence expected for an Fe-OH mode and its resonant scattering cross section which is comparable to that of the 493-cm-1 mode. This new species has a calculated Fe-O-Fe angle of approximately 113 degrees compared to approximately 138 degrees calculated for the Fe-O-Fe unit in unmodified protein B2. One possible explanation for the photoinduced vibrational mode is that a bridging solvent molecule has been inserted in place of a bridging carboxylate.

Escherichia coli↗

PubMed1988

The active site of hemerythrin as determined by X-ray absorption fine structure.

Extensive X-ray absorption fine structure measurements and analysis have been made on azidomet- and methemerythrin and on the native forms of oxy- and deoxyhemerythrin. Due to the availability of models that have been synthesized to mimic the active site of hemerythrin, it was possible to make a thorough assessment of the various errors in the structural parameters determined by the analysis. It is found that the largest source of error is the lack of complete transferability of amplitude and phase between the standards and hemerythrin. This is of particular importance in distinguishing the contributions of the second-shell low-Z atoms and, thus, has a substantial influence on the determination of the iron-iron distance. The internal consistencies of the various checks and a new formulation of error analysis for the structural parameters give us confidence in the structure determined for the active site. The main result is that as O2 is released from oxyhemerythrin, the mu-oxo bridge between the two iron atoms in the active site with an Fe-O distance of 1.8 A converts to a mu-hydroxo bridge in deoxyhemerythrin, expanding the Fe-O distance to 2.0 A. The Fe-Fe distance expands proportionally from 3.24 A in oxyhemerythrin to 3.57 A in deoxyhemerythrin so as to keep the Fe-O-Fe bridging angle approximately constant. These conclusions provide experimental support for the structures of oxy- and deoxyhemerythrin proposed previously on the basis of spectroscopic and preliminary X-ray crystallographic data.

Animals↗

PubMed1988

Resonance Raman spectroscopy of amicyanin, a blue copper protein from Paracoccus denitrificans.

The copper binding site of amicyanin from Paracoccus denitrificans has been examined by resonance Raman spectroscopy. The pattern of vibrational modes is clearly similar to those of the blue copper proteins azurin and plastocyanin. Intense resonance-enhanced peaks are observed at 377, 392, and 430 cm-1 as well as weaker overtones and combination bands in the high frequency region. Most of the peaks below 500 cm-1 shift 0.5-1.5 cm-1 to lower energy when the protein is exposed to D2O. Based on the pattern of conserved amino acids, the axial type EPR spectrum, and the resonance Raman spectrum, it is proposed that the copper binding site in amicyanin contains a Cu(II) ion in a distorted trigonal planar geometry with one cysteine and two histidine ligands and an axial methionine ligand at a considerably longer distance. Furthermore, the presence of multiple intense Raman peaks in the 400 cm-1 region which are sensitive to deuterium substitution leads to the conclusion that the Cu-S stretch is coupled with internal ligand vibrational modes and that the sulfur of the cysteine ligand is likely to be hydrogen-bonded to the polypeptide backbone.

Azurin↗

PubMed1988

Nitric oxide adducts of the binuclear iron site of hemerythrin: spectroscopy and reactivity.

Nitric oxide forms adducts with the binuclear iron site of hemerythrin (Hr) at [Fe(II),Fe(II)]deoxy and [Fe(II),Fe(III)]semimet oxidation levels. With deoxyHr our results establish that (i) NO binds reversibly, forming a complex which we label deoxyHrNO, (ii) NO forms a similar but distinct complex in the presence of fluoride, which we label deoxyHrFNO, (iii) NO is directly coordinated to one iron atom of the binuclear pair in these adducts, most likely in a bent end-on fashion, and (iv) the iron atoms in the binuclear sites of both deoxyHrNO and deoxyHrFNO are antiferromagnetically coupled, thereby generating unique electron paramagnetic resonance (EPR) detectable species. The novel EPR signal of deoxyHrNO (deoxyHrFNO) with g[[ = 2.77 (2.58) and g = 1.84 (1.80) is explained by the magnetic interaction of the Fe(II) (S' = 2) and [FeNO]7 (S = 3/2) centers observed by Mössbauer spectroscopy. Antiferromagnetic coupling leads to a ground state of Seff = 1/2. Analysis of the EPR parameters using the isotropic spin-exchange Hamiltonian, Hex = 2JS3/2.S2, and including zero-field splitting leads to a coupling constant, -J approximately 23 cm-1, for deoxyHrNO. The resonance Raman spectrum of deoxyHrNO shows features at 433 and 421 cm-1 that shift downward with 15N16O and that are assigned to stretching and bending modes, respectively, of the [FeNO]7 unit. Sensitivity of the bending mode to D2O suggests that bound NO participates in hydrogen bonding. We propose that the terminal oxygen atom of NO is hydrogen bonded to the proton of the mu-hydroxo bridge in the Fe-(OH)-Fe unit. A bent Fe-N-O geometry is supported by spectroscopic and structural comparisons to synthetic complexes and is consistent with a limiting [FeII,FeIIINO-] formulation for deoxyHrNO. Reversibility of NO binding to deoxyHr is demonstrated by bleaching of the optical and EPR spectra of deoxyHrNO upon additions of excess N3- or CNO-. DeoxyHrNO undergoes autoxidation under anaerobic conditions over the course of several hours. The product of this autoxidation appears to be an EPR-silent NO adduct of semimetHr. The formal one-electron oxidations of the binuclear iron site of deoxyHr by NO and by HNO2 can conceivably occur with no net change in charge on the iron site. In contrast, autoxidation of oxy- to metHr requires a change in net charge on the iron site, which may provide a kinetic barrier.

Animals↗

PubMed1988

New aerobactin-mediated iron uptake system in a septicemia-causing strain of Enterobacter cloacae.

Unlike the great majority of the aerobactin-producing enteric bacteria documented in the literature, Enterobacter cloacae EK33, isolated from a case of human neonatal meningitis, did not show any homology at the DNA level with the prototype aerobactin system encoded by the ColV-K30 plasmid. However, both the nuclear magnetic resonance spectrum and fast-atom bombardment mass spectrometry of the siderophore purified from EK33 confirmed its identity with aerobactin. Bioassay screening of a gene library of total DNA of EK33 led to the isolation of several aerobactin-positive clones. Under conditions of iron limitation, these clones expressed in Escherichia coli a protein of 72 kilodaltons that reacted with antiserum raised against the pColV-K30 74-kilodalton aerobactin receptor, while the original E. cloacae strain synthesized an 85-kilodalton protein which also cross-reacted with the antiserum. Restriction endonuclease analysis of the cloned DNA confirmed the structural differences between the two aerobactin genetic systems.

Conjugation, Genetic↗

PubMed1988

Involvement of oxo-bridged binuclear iron centers in oxygen transport, oxygen reduction, and oxygenation.

The occurrence of an oxo-bridged binuclear iron site is well-established for the oxygen transport protein, hemerythrin, and strongly implicated in ribonucleotide reductase, purple acid phosphatase, ferritin, and methane monooxygenase. Key identifying characteristics are an antiferromagnetic interaction between the two iron atoms, an Fe-O-Fe vibrational mode in the resonance Raman spectrum, and an S = 1/2 EPR signal upon one-electron reduction. In hemerythrin the oxo bridge serves as a hydrogen bond acceptor which stabilizes the bound hydroperoxide. In ribonucleotide reductase both the binuclear iron center and a protein tyrosine undergo oxidation in the presence of molecular oxygen, whereas in methane monooxygenase a binuclear iron moiety may activate O2 for substrate oxygenation.

Iron↗

PubMed1987

Hydrogen bonding of sulfur ligands in blue copper and iron-sulfur proteins: detection by resonance Raman spectroscopy.

The resonance Raman spectrum of the blue copper protein azurin from Alcaligenes denitrificans exhibits nine vibrational modes between 330 and 460 cm-1, seven of which shift 0.4-3.0 cm-1 to lower energy after incubation of the protein in D2O. These deuterium-dependent shifts have been previously ascribed to exchangeable protons on imidazole ligands [Nestor, L., Larrabee, J. A., Woolery, G., Reinhammar, B., & Spiro, T. G. (1984) Biochemistry 23, 1084] or to exchangeable protons on amide groups which are hydrogen bonded to the cysteine thiolate ligands (a feature common to all blue copper proteins of known structure). In order to distinguish between these two possibilities, a systematic investigation of Fe2S2(Cys)4-containing proteins was undertaken. Extensive hydrogen bonding between sulfur ligands and the polypeptide backbone had been observed in the crystal structure of ferredoxin from Spirulina platensis. The resonance Raman spectrum of this protein is typical of a chloroplast-type ferredoxin and exhibits deuterium-dependent shifts of -0.3 to -0.5 cm-1 in the Fe-S modes at 283, 367, and 394 cm-1 (assigned to the bridging sulfurs) and -0.6 to -0.8 cm-1 in the Fe-S modes at 328 and 341 cm-1 (assigned to the terminal cysteine thiolates). Considerably greater deuterium sensitivity is observed in the Raman spectra of spinach ferredoxin and bovine adrenodoxin, particularly for the symmetric stretching vibration of the Fe2S2 moiety at approximately 390 cm-1. This feature decreases by 0.8 and 1.1 cm-1, respectively, for the two oxidized proteins in D2O and by 1.8 cm-1 for reduced adrenodoxin in D2O.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗

PubMed1987

Identification of a hydroxide ligand at the iron center of ribonucleotide reductase by resonance Raman spectroscopy.

The resonance Raman spectrum of protein B2 of ribonucleotide reductase from Escherichia coli shows several features to its oxo-bridged binuclear iron center. A peak at 492 cm-1 is assigned to the symmetric stretch of the Fe-O-Fe moiety on the basis of its 13-cm-1 shift to lower energy upon 18O substitution. The 18O species shows an additional peak at 731 cm-1, which is a good candidate for the asymmetric stretch of the Fe-O-Fe moiety. Its exact location in the 16O species is obscured by the presence of a protein tryptophan vibration at 758 cm-1. A third resonance-enhanced peak at 598 cm-1 is identified as an Fe-OH vibration on the basis of its 24-cm-1 shift to lower energy in H2 18O, its 2-cm-1 shift to lower energy in D2O, and its pH-dependent intensity. A hydrogen-bonded mu-oxo bridge similar to that in hemerythrin is suggested by the unusually low frequency for the Fe-O-Fe symmetric stretch and the 3-cm-1 shift to higher energy of vs(Fe-O-Fe) in D2O. From the oxygen isotope dependence of vs(Fe-O-Fe), an Fe-O-Fe angle of 138 degrees can be calculated. This small angle suggests that the iron center consists of a tribridged core as in hemerythrin. A model for the binuclear iron center of ribonucleotide reductase is presented in which the hydroxide ligand sites provide an explanation for the half-of-sites reactivity of the enzyme.

Escherichia coli↗

PubMed1987

Induction of siderophore activity in Anabaena spp. and its moderation of copper toxicity.

Growth of Anabaena sp. strain 7120 (in the absence of chelators or added iron) was inhibited by the addition of 2.1 to 6.5 microM copper and was abolished by copper concentration of 10 microM or higher. When the copper was chelated to schizokinen (the siderophore produced by this organism in response to iron starvation), the toxic effects were eliminated. Analysis of culture filtrates showed that the cupric schizokinen remains in the medium, thereby lowering the amount of copper taken up by the cells. Although this organism actively transports ferric schizokinen, it apparently does not recognize the cupric complex. Thus, Anabaena sp. is protected from copper toxicity under conditions in which siderophore is being produced. For cells grown in low iron, the accumulation of extracellular schizokinen was observed to parallel cell growth and continue well into stationary phase. The actual iron status of the organism was monitored by using iron uptake velocity as an assay. Cultures grown on 0.1 microM added iron were found to be severely iron limited upon reaching stationary phase, thus explaining the continued production of schizokinen. These data show that the siderophore system in Anabaena spp. has developed primarily as a response to iron starvation and that additional functions such as alleviation of copper toxicity or allelopathic inhibition of other algal species are merely secondary benefits.

Copper↗

PubMed1986

Characterization of anguibactin, a novel siderophore from Vibrio anguillarum 775(pJM1).

Anguibactin, a siderophore produced by cells of Vibrio anguillarum 775 harboring the pJM1 plasmid, has now been isolated from the supernatants of iron-deficient cultures. This iron-reactive material was purified by adsorption onto an XAD-7 resin and subsequent gel filtration on a Sephadex LH-20 column. The resulting neutral compound produced an ion at m/z 348 in mass spectrometry and contained one sulfur, four oxygen, and four nitrogen atoms as determined by elemental analysis. Its strong UV absorbance and blue fluorescence were suggestive of a phenolic moiety. In colorimetric reactions anguibactin behaved like a catechol. The catechol assignment was supported by the appearance of a new absorption band at 510 nm in the ferric complex and by the appearance of peaks at 1,367, 1,447, 1,469, and 1,538 cm-1 in the resonance Raman spectrum. In addition, the infrared spectrum gave evidence of a secondary amide function, but no free carboxylic acid or hydroxamic acid groups were observed. A third iron-ligating group was suggested by the liberation of three protons during iron binding; mass spectrometry of the resulting material yielded a molecular ion characteristic of a 1:1 complex of ferric anguibactin. The purified anguibactin exhibited specific growth-promoting activity under iron-limiting conditions for a siderophore-deficient mutant of V. anguillarum 775(pJM1). A novel structure for anguibactin was indicated by the failure of a large number of known siderophores and synthetic chelators to yield a similar type of specific cross-feeding in the V. anguillarum bioassay.

Bacterial Outer Membrane Proteins↗

PubMed1985

Active site structures of deoxyhemerythrin and oxyhemerythrin.

The physiologically active forms of the nonheme-iron, oxygen-transport protein hemerythrin have been studied by x-ray crystallographic techniques. At 3.9-A resolution, a difference electron-density map between the deoxy form and met form (methemerythrin) of the protein suggests only small differences in the binuclear iron complexes. The coordination of the iron atoms appears to be the same in both the deoxy and met forms, one iron of the complexes being pentacoordinate, the other iron being hexacoordinate. The iron atoms appear to be somewhat farther apart in the deoxy form. A 2.2-A resolution study of oxyhemerythrin shows that dioxygen binds to one iron atom--the pentacoordinate one in the met form of the protein, the same binding site found for azide in azidomethemerythrin.

Animals↗

PubMed1984

Sulfide-bridged derivatives of the binuclear iron site of hemerythrin at both met and semi-met oxidation levels.

Exposure of methemerythrin (metHr) to S2- under anaerobic conditions results in a one-electron reduction to the semi-met level and replacement of the mu-oxo bridge between the irons with a single sulfide. The sulfide bridge is maintained upon ferricyanide oxidation of semi-metsulfide to metsulfide hemerythrin and upon subsequent dithionite or S2- reduction back to the semi-met level. Chemical analyses show that metsulfideHr contains one S2- per two Fe. The single quadrupole doublet (delta = 0.50 mm/s; delta Eq = 0.99 mm/s) in the Mössbauer spectrum is consistent with a bridging sulfide geometry. The optical and resonance Raman spectra of metsulfideHr are reminiscent of the [2Fe-2S] iron-sulfur proteins. The optical spectrum exhibits multiple S2----Fe(III) charge-transfer transitions between 400 and 600 nm. The resonance Raman spectrum reveals a series of overtones and combinations of the 431-cm-1 Fe-S-Fe symmetric vibration and the 327-cm-1 asymmetric vibration. The relative energies of the symmetric and asymmetric modes are characteristic of a sulfur-bridged system with a bridge angle of approximately 80 degrees. MetsulfideHr decomposes over several hours in air and over several days in the absence of O2 to metHr and semi-metsulfideHr, respectively. Unlike metHr and semi-metHr, neither the metsulfide nor the semi-metsulfide derivatives form stable adducts with anions such as azide or cyanide. Sulfide bridging confers new properties on the binuclear iron center that are of interest to an understanding of the chemistry of hemerythrin and also of the [2Fe-2S] iron-sulfur proteins.

Animals↗

PubMed1984

Hydroxide ion binding to methemerythrin. An investigation by resonance Raman and difference spectroscopy.

The pH dependence for the interconversion of the acid and base forms of methemerythrin from Themiste dyscritum was investigated by difference spectroscopy. A new technique was designed to be able to study mixtures without knowledge of extinction coefficients or exact protein concentrations. The resultant pKa value of 8.4 proved that T. dyscritum hemerythrin crystals used for previous X-ray crystallographic studies at pH less than or equal to 6.5 were in the acid form. Since this material contains a 5-coordinate iron atom with no evidence of a ligated water molecule, it is more appropriately referred to as methemerythrin than aquomethemerythrin. The presence of an iron-bound hydroxide in the base form of methemerythrin was verified by resonance Raman spectroscopy for both T. dyscritum and Phascolopsis gouldii. At pH greater than 9, the protein from either species exhibited a new feature at 490 cm-1 that shifted to 518 cm-1 in D2O and was assigned to a coupled Fe-OH stretching and O-H bending vibration. Thus, hydroxomethemerythrin is the correct designation for the base form of the protein. The other resonance-enhanced vibration, the Fe-O-Fe symmetric stretch, was observed at 506 cm-1 in hydroxomethemerythrin and at 511 cm-1 in methemerythrin and was unaffected by deuteration. Addition of perchlorate to methemerythrin had no effect on the Raman spectrum, despite its known role in stabilizing the met form relative to the hydroxomet form.

Animals↗

PubMed1983

Resonance Raman spectra of copper(II)-substituted liver alcohol dehydrogenase: a type 1 copper analogue.

Liver alcohol dehydrogenase (LADH) with copper in place of the catalytic zinc has recently been proposed to contain a type 1 site analogous to that in "blue" copper proteins. Resonance Raman spectra for the copper-substituted enzyme, Cu(II) X LADH, and its binary complexes with reduced nicotinamide adenine dinucleotide (NADH) and pyrazole support this viewpoint. These spectra have two dominant features: a sharp peak at approximately 415 cm-1, which is believed to be associated with vibration of the single histidine ligand, and a broader, asymmetric band at approximately 350 cm-1, whose components are assigned predominantly to vibrational modes of the two cysteinate ligands. The high frequency of these transitions, which is reminiscent of the blue copper proteins, is ascribed to the tetrahedral nature of the metal site that produces unusually short Cu-S bonds and coupled vibrational modes. Solvent exchange with H218O reveals no contribution to the resonance Raman spectrum of the water molecule, which is a metal ligand in free Cu(II) X LADH; however, the spectrum of the binary complex with pyrazole has several new peaks attributable, in part, to pyrazole ligation. The strong similarity among the vibrational spectra demonstrates that the Cu(II) environment in alcohol dehydrogenase maintains its near-tetrahedral geometry in the various enzyme derivatives. The resonance Raman spectrum of Ni(II) X LADH is close to that of Cu(II) X LADH and suggests a similar tetrahedral site. The Raman spectra presented here together with available optical and EPR data indicate that Cu(II) X LADH belongs to the type 1 copper classification and, thus, can provide new insights into this unusual coordination geometry.

Alcohol Dehydrogenase↗

PubMed1983

Siderophore-mediated iron uptake in different strains of Anabaena sp.

Anabaena sp. strain 6411, which produces the dihydroxamate siderophore schizokinen to facilitate iron uptake, is also capable of using the related siderophore aerobactin. The two siderophores compete for the same iron transport system, but there is a markedly higher affinity for ferric schizokinen than for ferric aerobactin. The trihydroxamate siderophore ferrioxamine B is far less effective as an iron donor in this organism. Anabaena sp. strain 7120 appears to be closely related to strain 6411. It synthesizes schizokinen as its major siderophore and shows rates of iron uptake from ferric schizokinen, ferric aerobactin, and ferrioxamine B which are similar to those observed with strain 6411. Anabaena cylindrica Lemm. 7122 and 1611, on the other hand, differ from strain 6411. In contrast to schizokinen, the hydroxamate which they produce in response to iron starvation cannot be extracted with water from the organic layer and does not support the growth of the siderophore auxotroph Arthrobacter flavescens JG-9. Strain 7122 can use its endogenous siderophore or schizokinen to promote iron uptake, but at 50-fold-lower rates than are observed with Anabaena sp. strain 6411 or 7120.

Biological Transport↗

PubMed1982

Raman spectral evidence for a mu-oxo bridge in the binuclear iron center of ribonucleotide reductase.

The Raman spectrum of the B2 subunit of Escherichia coli ribonucleotide reductase shows a peak at 496 cm-1 that appears to be in resonance with the 370-nm electronic transition of the binuclear iron center in both the native and radical-free forms of the protein. Exposure of the protein to H218O causes the peak to shift to 481 cm-1, indicating that the vibrational mode is due to an Fe-O moiety in which the oxygen can exchange with solvent. The rate of oxygen exchange (kobsd = 8.3 x 10-4 s-1) is consistent with a mu-oxo-bridged structure. Protonation of the oxygen is unlikely since the Fe-O vibration fails to shift to lower frequency in D2O. Instead, there is a gradual increase in the vibrational frequency with time to a maximum value of 502 cm-1 after 3 h in 70% with time to a maximum value of 602 cm-1 after 3 h in 70% D2O. Apparently, the deuteration of successive protein functional groups causes a slight alteration in the structure of the binuclear iron center. The resonance Raman characteristics of the Fe-O-Fe group in protein B2 are similar to those previously reported for the mu-oxo-bridged binuclear iron center in hemerythrin. A further similarity between the two proteins is the high degree of alpha-helical content. Circular dichroism measurements place this value at approximately 60% for the B2 subunit of ribonucleotide reductase.

Circular Dichroism↗

PubMed1982

Active transport of ferric schizokinen in Anabaena sp.

The cyanobacterium Anabaena sp. strain ATCC 27898 was found to utilize the siderophore schizokinen to accumulate iron from the environment. This organism had previously been shown to produce schizokinen under low-iron conditions, and we observed that the iron-transport capability is also increased in response to iron limitation. Uptake activity was specific for ferric schizokinen displayed kinetics typical of a protein-mediated process with an apparent Km of 0.04 microM and saturation at high concentrations of substrate. Light-driven transport was blocked by uncouplers and by ATPase inhibitors. Transport in dark-adapted cells was additionally blocked by inhibitors of respiration. We conclude that ATP serves as an energy source for the cellular uptake of ferric schizokinen.

Biological Transport, Active↗