Proton-linked subunit heterogeneity in ferrous nitrosylated human adult hemoglobin: an EPR study

The effect of pH on the X-band electron paramagnetic resonance (EPR) spectrum of ferrous nitrosylated human adult tetrameric hemoglobin (HbNO) as well as of ferrous nitrosylated monomeric alpha- and beta-chains has been investigated, at -163 degrees C. At pH 7.3, the X-band EPR spectrum of tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains displays a rhombic shape. Lowering the pH from 7.3 to 3.0, tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains undergo a transition towards a species characterized by a X-band EPR spectrum with a three-line splitting centered at 334mT. These pH-dependent spectroscopic changes may be taken as indicative of the cleavage, or the severe weakening, of the proximal HisF8-Fe bond. In tetrameric HbNO, the pH-dependent spectroscopic changes depend on the acid-base equilibrium of two apparent ionizing groups with pK(a) values of 5.8 and 3.8. By contrast, the pH-dependent spectroscopic changes occurring in ferrous nitrosylated monomeric alpha- and beta-chains depend on the acid-base equilibrium of one apparent ionizing group with pK(a) values of 4.8 and 4.7, respectively. The different pK(a) values for the proton-linked spectroscopic transition(s) of tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains suggest that the quaternary assembly drastically affects the strength of the proximal HisF8-Fe bond in both subunits. This probably reflects a 'quaternary effect', i.e., structural changes in both subunits upon tetrameric assembly, which is associated to a relevant variation of functional properties (i.e., proton affinity).     

Nitrosylation of rabbit ferrous heme-hemopexin

Hemopexin (HPX) serves as a trap for toxic plasma heme, ensuring its complete clearance by transportation to the liver. Moreover, HPX-heme has been postulated to play a key role in the homeostasis of nitric oxide (NO). Here, the thermodynamics for NO binding to rabbit ferrous HPX-heme as well as the EPR and optical absorption spectroscopic properties of rabbit ferrous nitrosylated HPX-heme (HPX-heme-NO) are reported. The value of the dissociation equilibrium constant for NO binding to rabbit ferrous HPX-heme (i.e., H) is (1.4±0.2)×10^–7 M, at pH 7.0 and 10.0 °C; the value of H is unaffected by sodium chloride. At pH 7.0, rabbit ferrous HPX-heme-NO is a six-coordinate heme-iron species, characterized by an X-band EPR spectrum with an axial geometry and by =146 mM^–1 cm^–1 at 419 nm. At pH 4.0, rabbit ferrous HPX-heme-NO is a five-coordinate heme-iron species, characterized by an X-band EPR spectrum with three-line splitting centered at 334 mT and by =74 mM^–1 cm^–1 at 387 nm. The pK_a value of the reversible pH-induced six- to five-coordinate spectroscopic transition is 4.8±0.1 in the absence of sodium chloride and 4.3±0.1 in the presence of 1.5×10^–1 M sodium chloride. This result is in agreement with the effect of sodium chloride on rabbit HPX-heme stability. The present data have been analyzed in parallel with those of a related heme model compound and heme-protein systems.     

Effect of bezafibrate and clofibrate on the heme-iron geometry of ferrous nitrosylated heme-human serum albumin: an EPR study

The effect of bezafibrate (BZF) and clofibrate (CF), two therapeutic drugs displaying anticoagulant and antihyperlipoproteinemic activities, on the EPR-spectroscopic properties of ferrous nitrosylated heme-human serum albumin (HSA-heme-NO) has been investigated. In the absence of BZF and CF, HSA-heme-NO is a five-coordinate heme-iron system, characterised by an X-band EPR spectrum with a three-line splitting in the high magnetic field region. Addition of BZF and CF to HSA-heme-NO induced the transition towards a six-coordinate heme-iron species characterised by an X-band EPR spectrum with an axial geometry. These data indicate that HSA-heme-NO is a five-coordinate heme-iron system, BZF and CF acting as allosteric effectors, and show that the primary heme binding site and the CF cleft of HSA are conformationally-linked, regardless of their different location.     

The heme-iron geometry of ferrous nitrosylated heme-serum lipoproteins,hemopexin, and albumin: a comparative EPR study

Serum high and low density lipoproteins, albumin, and hemopexin (HDL, LDL, SA, and HPX, respectively) serve as traps of toxic plasma heme and participate in its complete clearance by transportation to the liver. Moreover, SA-(heme) and HPX-heme have been proposed to facilitate NO scavenging in vivo. Here, the EPR-spectroscopic properties of ferrous nitrosylated heme-human high and low density lipoproteins (HDL-heme-NO and LDL-heme-NO, respectively) as well as of ferrous nitrosylated heme-rabbit serum hemopexin (HPX-heme-NO) are reported and analyzed in parallel with those of ferrous nitrosylated heme-human serum albumin (SA-heme-NO). HDL-heme-NO and LDL-heme-NO as well as SA-heme-NO, in the absence of allosteric effectors (i.e., N-form), are five-coordinate heme-iron species, characterized by the three-line splitting observed in the high magnetic field region of the X-band EPR spectrum. On the other hand, SA-heme-NO, in the presence of drugs (i.e., B-form), and HPX-heme-NO are six-coordinate heme-iron species, characterized by an X-band EPR spectrum with an axial geometry. The heme-iron coordination state of HDL-heme-NO, LDL-heme-NO, SA-heme-NO, and HPX-heme-NO is in keeping with values of ferric heme dissociation rate constants which decrease in the following order: LDL>HDL>SA>HPX. Altogether, these observations suggest that HPX displays a cleft much more suitable for heme binding than other heme-carriers.     

The NAD-linked soluble hydrogenase from Alcaligenes eutrophus H16: detection and characterization of EPR signals deriving from nickel and flavin

 In this study we confirmed the previous observation that the cytoplasmic NAD-linked hydrogenase of Alcaligenes eutrophus H16 is EPR-silent in the oxidized state. We also demonstrated the presence of significant Ni-EPR signals when the enzyme was either reduced with the natural electron carrier NADH (5–10 mM) or carefully titrated with sodium dithionite to an intermediate, narrow redox potential range (–280 to –350 mV). Reduction with NADH under argon atmosphere led to a complex EPR spectrum at 80 K with g values at 2.28, 2.20, 2.14, 2.10, 2.05, 2.01 and 2.00. This spectrum could be differentiated by special light/dark treatments into three distinct signals: (1) the "classical" Ni-C signal with g values at 2.20, 2.14 and 2.01, observed with many hydrogenases in the reduced, active state; (2) the light-induced signal (Ni-L) with g values at 2.28, 2.10 and 2.05 and (3) a flavin radical (FMN semiquinone) signal at g = 2.00. The assignment of the Ni-EPR signal was clearly confirmed by EPR spectra of hydrogenase labeled with ⁶¹Ni (nuclear spin I = 3/2) yielding a broadening of the Ni spectra at all g values and a resolved ⁶¹Ni hyperfine splitting into four lines of the low field edge in the case of the light-induced Ni-EPR signal. The redox potentials determined at pH 7.0 for the described redox components were: for FMN –170 mV (midpoint potential, E_m, for appearance), –200 mV (EPR signal intensity maximum) and –230 mV (E_m for disappearance); for the Ni centre (Ni-C), –290 mV (E_m for appearance), –305 mV (signal intensity maximum) and –325 mV (E_m for disappearance). Exposure of the NADH-reduced hydrogenase to carbon monoxide led to an apparent Ni-CO species indicated by a novel rhombic EPR signal with g values at 2.35, 2.08 and 2.01. Received: 19 July 1995 / Accepted: 10 September 1995     

Characterization of cytochrome c 6 from the cyanobacterium Anabaena PCC 7119

 A soluble monoheme c–type cytochrome c ₆ has been isolated from the cyanobacterium Anabaena PCC 7119. It is a basic protein, with a molecular mass of 9.7 kDa, which accepts electrons from Anabaena ferredoxin in the ferredoxin-NADP⁺reductase-dependent NADPH cytochrome c reductase activity assay. The turnover of the reaction has an optimum pH at 7.5. Flavodoxin can also replace ferredoxin in this assay, but with only 20% efficiency. Plastocyanin from Anabaena PCC 7119, as well as the c ₆ cytochromes from the green algae Chlorella fusca and Monoraphidium braunii are also shown to accept electrons from Anabaena ferredoxin. The reduction potential of cytochrome c ₆ at pH 6.7 was determined to be 338 mV and is pH dependent, with pK _a ^ox=8.4±0.1 and pK _a ^red≈9.5. The ferric and ferrous cytochrome forms and their pH equilibria have been studied using visible, EPR and ¹H-NMR spectroscopies. The amino acid sequence and the visible and NMR spectroscopic data indicate that the heme iron has a methionine-histidine axial coordination in the pH range 5–11. However, the EPR data for the ferricytochrome are complex and show that in this pH range five distinct forms are present. Between pH 5 and 9 the spectrum is dominated by two rhombic species, with g–values at 2.94, 2.29, 1.43 and at 2.84, 2.34, 1.56, which interconvert with a pK _a of 8.4. The NMR data also show a main interconversion between two cytochrome forms at this pH, which coincides with that determined from the pH dependence of the reduction potential. Both these forms were associated with a methionine-histidine heme-iron coordination by correlation with the visible and NMR spectral data, although having crystal field parameters atypical for this type of coordination. Anabaena cytochrome c ₆ is one more example of a heme protein for which the widely used crystal field analysis of the EPR data (truth diagram) fails to unequivocally determine the type of heme-iron ligation. Received: 17 May 1996 / Accepted: 13 January 1997     

EPR evidence of two structurally different diferric sites in Mycobacterium tuberculosis R2-2 ribonucleotide reductase protein

Mixed-valent species were generated in the diiron site of active (with tyrosyl free radical) and met (without radical) forms of protein R2-2 in a class Ib ribonucleotide reductase from Mycobacterium tuberculosis by low temperature reduction (γ-irradiation) at 77 K. The primary mixed-valent EPR signal is a mixture of two components with axial symmetry and g_av<2.0, observable at temperatures up to 77 K, and assigned to antiferromagnetically coupled high spin ferric/ferrous sites. The two components in the primary EPR signal can be explained by the existence of two structurally distinct μ-oxo-bridged diferric centers, possibly related to structural heterogeneity around the iron site, and/or different properties of the two polypeptide chains in the homodimeric protein after the radical reconstitution reaction. Annealing of the irradiated R2-2 samples to 143 K transforms the primary EPR signal into a rhombic spectrum characterized by g_av<1.8 and observable only below 25 K. This spectrum is assigned to a partially relaxed form with a μ-hydroxo-bridge. Further annealing at 228 K produces a new complex rhombic EPR spectrum composed of at least two components. An identical EPR spectrum was observed and found to be stable upon chemical reduction of Mycobacterium tuberculosis RNR R2-2 at 293 K by dithionite.     

Different structural effects of allosteric modulators on subunits of tetrameric ferrous nitrosylated human hemoglobin: an EPR spectroscopic study

 The X-band EPR spectroscopic features of the ferrous nitrosylated derivative of α(Fe)₂β(Co)₂ and of α(Co)₂β(Fe)₂ metal hybrids of human hemoglobin (Hb) have been investigated at pH 7.0 and analyzed in parallel with those of the native nitrosylated tetramer (HbNO). The effect of 2,3-biphosphoglycerate (BPG), inositol hexakisphosphate (IHP) and bezafibrate (BZF) has been investigated in order to understand the perturbations induced on α and β subunits in the tetramer by the binding of allosteric effectors. A large perturbation is observed in both subunits upon BZF binding, while in the case of IHP only α-chains are affected; on the other hand, BPG leaves both chains essentially unperturbed. Thus, different binding modes of allosteric effectors to HbNO may occur, and the simultaneous addition of two effector molecules, namely BPG and BZF or IHP and BZF to HbNO, brings about different alterations of the X-band EPR spectroscopic properties. This behavior indicates that the intramolecular communication pathway(s) between the heme and the binding pockets of the heterotropic ligands (i.e., IHP and BZF, or BPG and BZF) are different, leading to distinct structural perturbations. Received: 19 September 1997 / Accepted: 16 December 1997     

The acid-alkaline transition of a sea turtle myoglobin: coexistence at high pH of high- and low-spin forms

The microenvironment of the iron in a sea turtle Dermochelys coriacea myoglobin is studied using the spectroscopic techniques EPR and optical absorption. Optical absorption spectra in the visible region suggest a great homology between turtle Mb and other myoglobins, such as those from whale, human and elephant. The pK of the acid-alkaline transition is 8.4 slightly lower than the pK of whale and equal to that of elephant myoglobin. The EPR spectrum at pH 7.0 is characteristic of a high-spin configuration with axial symmetry (gx = gy = 5.95). At higher pH, this signal changes in a way different from that observed for whale myoglobin. We observe for turtle Mb both the formation of a low-spin configuration with rhombic symmetry (gx = 2.56, gy = 2.20, gz = 1.90) and of a high-spin species with rhombic distortion (gx = 6.79, gy = 5.18, gz = 2.12). This suggests a lowering of symmetry at the haem, so that now the x and y directions are no more equivalent. This can be explained by amino acid substitution at the distal positions of haem or to off-axial positioning of distal residues. The coexistence at high pH (pH 11.0) of these two spin forms could be explained by the existence of two protein conformations, in which the crystal field splitting factor, delta, and the electron exchange energy are of the same order, allowing the presence of different configurations simultaneously. The presence of different kinds of haem is ruled out by the experiments with nitrosyl turtle Mb and turtle Mb-F showing spectra very similar to those of whale myoglobin. The pk of the acid-alkaline transition, 8.5, obtained from EPR spectra, agrees very well with results from optical absorption.     

EPR properties of mixed-valent μ-oxo and μ-hydroxo dinuclear iron complexes produced by radiolytic reduction at 77 K

 Radiolytic reduction at 77 K of oxo-/hydroxo-bridged dinuclear iron(III) complexes in frozen solutions forms kinetically stabilized, mixed-valent species in high yields that model the mixed-valent sites of non-heme, diiron proteins. The mixed-valent species trapped at 77 K retain ligation geometry similar to the initial diferric clusters. The shapes of the mixed-valent EPR signals depend strongly on the bridging ligands. Spectra of the Fe(II)OFe(III) species reveal an S=1/2 ground state with small g-anisotropy as characterized by the uniaxial component (g _z –g _av /2<0.03) observable at temperatures as high as ∼100 K. In contrast, hydroxo-bridged mixed-valent species are characterized by large g-anisotropy (g _z –g _av /2>0.03) and are observable only below 30 K. Annealing at higher temperatures causes structural relaxation and changes in the EPR characteristics. EPR spectral properties allow the oxo- and hydroxo-bridged, mixed-valent diiron centers to be distinguished from each other and can help characterize the structure of mixed-valent centers in proteins. Received: 27 June 1998 / Accepted: 25 February 1999     

HisE11 and HisF8 Provide Bis-histidyl Heme Hexa-coordination in the Globin Domain of Geobacter sulfurreducens Globin-coupled Sensor

Among heme-based sensors, recent phylogenomic and sequence analyses have identified 34 globin coupled sensors (GCS), to which an aerotactic or gene-regulating function has been tentatively ascribed. Here, the structural and biochemical characterization of the globin domain of the GCS from Geobacter sulfurreducens (GsGCS¹⁶²) is reported. A combination of X-ray crystallography (crystal structure at 1.5 Å resolution), UV-vis and resonance Raman spectroscopy reveals the ferric GsGCS¹⁶² as an example of bis-histidyl hexa-coordinated GCS. In contrast to the known hexa-coordinated globins, the distal heme-coordination in ferric GsGCS¹⁶² is provided by a His residue unexpectedly located at the E11 topological site. Furthermore, UV-vis and resonance Raman spectroscopy indicated that ferrous deoxygenated GsGCS¹⁶² is a penta-/hexa-coordinated mixture, and the heme hexa-to-penta-coordination transition does not represent a rate-limiting step for carbonylation kinetics. Lastly, electron paramagnetic resonance indicates that ferrous nitrosylated GsGCS¹⁶² is a penta-coordinated species, where the proximal HisF8-Fe bond is severed.     

Yeast cytochrome c peroxidase. Coordination and spin states of heme prosthetic group

Electronic absorption and electron paramagnetic resonance (EPR) spectroscopic examinations revealed that a freshly prepared cytochrome c peroxidase (CCP) contains a penta-coordinated high spin ferric protoheme group. The penta-coordinated high spin state of fresh CCP is maintained in a remarkably wide range of pH (4-8). The freezing of fresh CCP induces the reversible coordination of an internal strong field ligand to the heme iron to form a hexa-coordinated low spin compound, which shows EPR extrema at gx = 2.70, gy = 2.20 and gz = 1.78. In the presence of glycerol the freezing-induced artifacts are eliminated and the fresh enzyme exhibits an EPR spectrum of rhombically distorted axial symmetry with EPR extrema at gx = 6.4, gy = 5.3, and gz = 1.97 at 10 K, characteristic of the penta-coordinated high spin enzyme. Upon aging CCP is converted to a hexa-coordinated high spin state due to the coordination of an internal weak field ligand to the heme iron. This conversion is accelerated at acidic pH values, and its reversibility varies from fully reversible to irreversible depending on the degree of enzyme aging. The aging-induced hexa-coordinated CCP is unreactive with hydrogen peroxide and exhibits an EPR spectrum of purely axial symmetry with extrema at g = 6 and g = 2 and an electronic absorption spectrum with an intensified Soret band at 408 nm (epsilon 408 nm = 120 mM-1 cm-1) and a blue-shifted charge-transfer band at 620 nm. Spectroscopic properties of different coordination and spin states of fresh and aged CCPs are compiled in order to formulate a generalized spectroscopic characterization of penta- and hexa-coordinated high spin ferric hemoproteins.     

Access of ligands to the ferric center in lipoxygenase-1.

A form of ferric lipoxygenase-1 has been isolated that gives an EPR spectrum that is dominated by a species of intermediate rhombicity (E/D = 0.065). This species is obtained in the presence of a number of buffers of high concentration and in the absence of fatty acid byproducts of the iron oxidation. The species is unstable over a period of one day with respect to symmetry of the iron. The EPR lineshapes of the unstable species are highly sensitive to the anionic composition of the buffer and to the addition of neutral ligands. These results suggest that newly formed ferric lipoxygenase has weak affinity for a number of ligands. Affinity of charged ligands for the iron center may provide a mechanism for charge compensation as the iron center alternates between ferric and ferrous in the catalytic cycle. We use spectral simulation to evaluate quantitatively the interaction of the ferric center with ligands and also show that a transition in the middle Kramers doublet makes a significant contribution to the EPR spectrum of the more rhombic species.     

EPR evidence of two structurally different diferric sites in Mycobacterium tuberculosis R2-2 ribonucleotide reductase protein

Mixed-valent species were generated in the diiron site of active (with tyrosyl free radical) and met (without radical) forms of protein R2-2 in a class Ib ribonucleotide reductase from Mycobacterium tuberculosis by low temperature reduction (γ-irradiation) at 77 K. The primary mixed-valent EPR signal is a mixture of two components with axial symmetry and g_av<2.0, observable at temperatures up to 77 K, and assigned to antiferromagnetically coupled high spin ferric/ferrous sites. The two components in the primary EPR signal can be explained by the existence of two structurally distinct μ-oxo-bridged diferric centers, possibly related to structural heterogeneity around the iron site, and/or different properties of the two polypeptide chains in the homodimeric protein after the radical reconstitution reaction. Annealing of the irradiated R2-2 samples to 143 K transforms the primary EPR signal into a rhombic spectrum characterized by g_av<1.8 and observable only below 25 K. This spectrum is assigned to a partially relaxed form with a μ-hydroxo-bridge. Further annealing at 228 K produces a new complex rhombic EPR spectrum composed of at least two components. An identical EPR spectrum was observed and found to be stable upon chemical reduction of Mycobacterium tuberculosis RNR R2-2 at 293 K by dithionite.     

Direct observation of multiple protonation states in recombinant human purple acid phosphatase

To date, most spectroscopic studies on mammalian purple acid phosphatases (PAPs) have been performed at a single pH, typically pH 5. The catalytic activity of these enzymes is, however, pH dependent, with optimal pH values of 5.5–6.2 (depending on the form). For example, the pH optimum of PAPs isolated as single polypeptides is around pH 5.5, which is substantially lower that of proteolytically cleaved PAPs (ca. pH 6.2). In addition, the catalytic activity of single polypeptide PAPs at their optimal pH values is four to fivefold lower than that of the proteolytically cleaved enzymes. In order to elucidate the chemical basis for the pH dependence of these enzymes, the spectroscopic properties of both the single polypeptide and proteolytically cleaved forms of recombinant human PAP (recHPAP) and their complexes with inhibitory anions have been examined over the pH range 4 to 8. The EPR spectra of both forms of recHPAP are pH dependent and show the presence of three species: an inactive low pH form (pH<pK _a,1), an active form (pK _a,1<pH<pK _a,2), and an inactive high pH form (pH>pK _a,2). The pK _a,1 values observed by EPR for the single polypeptide and proteolytically cleaved forms are similar to those previously observed in kinetics studies. The spectroscopic properties of the enzyme–phosphate complex (which should mimic the enzyme–substrate complex), the enzyme–fluoride complex, and the enzyme–fluoride–phosphate complex (which should mimic the ternary enzyme–substrate–hydroxide complex) were also examined. EPR spectra show that phosphate binds to the diiron center of the proteolytically cleaved form of the enzyme, but not to that of the single polypeptide form. EPR spectra also show that fluoride binds only to the low pH form of the enzymes, in which it presumably replaces a coordinated water molecule. The binding of fluoride and phosphate to form a ternary complex appears to be cooperative.Electronic Supplementary Material Supplementary material is available for this article at     

Spectroscopic and electrochemical properties of two azurins (Az-iso1 and Az-iso2) from the obligate methylotroph Methylomonas sp. strain J and the structure of novel Az-iso2

Methylomonas sp. strain J gives rise to two azurins (Az-iso1 and Az-iso2) with methylamine dehydrogenase (MADH-Mj). The intense blue bands characteristic of Az-iso1 and Az-iso2 are observed at 621 and 616 nm in the visible absorption spectra respectively, being revealed at 620−630 nm in those of usual azurins. The EPR signal of Az-iso1, similar to usual azurins, shows axial symmetry, while the axial EPR signal of Az-iso2 involves a slightly rhombic character. The half-wave potentials (E _1/2) of the two azurins and the intermolecular electron-transfer rate constants (k _ET) from MADH-Mj to each azurin were determined by cyclic voltammetry. The E _1/2 values of Az-iso1 and Az-iso2 are +321 and +278 mV vs NHE at pH 7.0, respectively. The k _ET value of Az-iso2 is larger than that of Az-iso1 by a factor of 5. However, the electron-transfer rate of Az-iso2 is interestingly slower than those of the azurins from a denitrifying bacterium, Alcaligenes xylosoxidans NCIB 11015, and the amicyanin from a different methylotroph, Methylobacterium extorquens AM1. The structure of Az-iso2 has been determined and refined against 1.6 Å X-ray diffraction data. The whole structure of Az-iso2 is quite similar to those of azurins reported already. The Cu(II) site of Az-iso2 is a distorted trigonal bipyramidal geometry like those of other azurins, but some of the Cu-ligand distances and ligand-Cu-ligand bond angle parameters are slightly different. These findings suggest that Az-iso2 is a novel azurin and perhaps functions as an electron acceptor for MADH. Received: 23 February 1999 / Accepted: 9 September 1999     

The solution structure of a monomeric, reduced form of human copper,zinc superoxide dismutase bearing the same charge as the native protein

2 , has RMSD values with respect to the average structure of 0.94 ± 0.14 Ų and 1.50 ± 0.14 Ų for the backbone and the heavy atoms, respectively. The overall folding, which includes the classical eight-stranded Greek-key β-barrel and a short α-helix, is very close to that of the previously characterized monomeric mutant E133QM2SOD and to that of wild-type SOD. The region involved in the subunit-subunit interactions in the dimeric protein is confirmed to be disordered in the monomeric species. It is also observed that a sizable rearrangement of the charged groups of the electrostatic loop and of Arg143 takes place in the monomeric species. The width of the active site channel, both at its entrance and at the bottleneck of the active site, is discussed in the light of the influence on the enzymatic activity and the latter with respect to the overall charge. It is also confirmed that the NH proton of His63 shields the Cu(I) from the bulk solvent, thus supporting the suggestion that superoxide may interact with the reduced metal ion in an outer-sphere fashion. Received: 14 May 1999 / Accepted: 17 September 1999     

Orientation-selected ENDOR of the active center in Chromatium vinosum [NiFe] hydrogenase in the oxidized "ready" state

 Electron nuclear double resonance (ENDOR) was applied to study the active site of the oxidized "ready" state, Ni_r, in the [NiFe] hydrogenase of Chromatium vinosum. The magnetic field dependence of the EPR was used to select specific subsets of molecules contributing to the ENDOR response by stepping through the EPR envelope. Three hyperfine couplings could be clearly followed over the complete field range. Two protons, H1 and H2, display a very similar large isotropic coupling of 12.5 and 12.6 MHz, respectively. Their dipolar coupling is small (2.1 and 1.4 MHz, respectively). A third proton, H3, exhibits a small isotropic coupling of 0.5 MHz and a larger anisotropic contribution of 3.5 MHz. Based on a comparison with structural data obtained from X-ray crystallography of single crystals of hydrogenases from Desulfovibrio gigas and D. vulgaris and the known g-tensor orientation of Ni_r, an assignment of the ¹H hyperfine couplings could be achieved. H1 and H2 were assigned to the β-CH₂ protons of the bridging cysteine Cys533 and H3 could belong to a β-CH₂ proton of Cys68 or to a protonated cysteine (-SH) of Cys68 or Cys530. Received: 26 November 1998 / Accepted: 1 April 1999     

Effect of ibuprofen and warfarin on the allosteric properties of haem-human serum albumin. A spectroscopic study.

Haem binding to human serum albumin (HSA) endows the protein with peculiar spectroscopic properties. Here, the effect of ibuprofen and warfarin on the spectroscopic properties of ferric haem-human serum albumin (ferric HSA-haem) and of ferrous nitrosylated haem-human serum albumin (ferrous HSA-haem-NO) is reported. Ferric HSA-haem is hexa-coordinated, the haem-iron atom being bonded to His105 and Tyr148. Upon drug binding to the warfarin primary site, the displacement of water molecules--buried in close proximity to the haem binding pocket--induces perturbation of the electronic absorbance properties of the chromophore without affecting the coordination number or the spin state of the haem-iron, and the quenching of the 1H-NMR relaxivity. Values of Kd for ibuprofen and warfarin binding to the warfarin primary site of ferric HSA-haem, corresponding to the ibuprofen secondary cleft, are 5.4 +/- 1.1 x 10(-4) m and 2.1 +/- 0.4 x 10(-5) m, respectively. The affinity of ibuprofen and warfarin for the warfarin primary cleft of ferric HSA-haem is lower than that reported for drug binding to haem-free HSA. Accordingly, the Kd value for haem binding to HSA increases from 1.3 +/- 0.2 x 10(-8) m in the absence of drugs to 1.5 +/- 0.2 x 10(-7) m in the presence of ibuprofen and warfarin. Ferrous HSA-haem-NO is a five-coordinated haem-iron system. Drug binding to the warfarin primary site of ferrous HSA-haem-NO induces the transition towards the six-coordinated haem-iron species, the haem-iron atom being bonded to His105. Remarkably, the ibuprofen primary cleft appears to be functionally and spectroscopically uncoupled from the haem site of HSA. Present results represent a clear-cut evidence for the drug-induced shift of allosteric equilibrium(a) of HSA.     

Bioactive metabolites from <Emphasis Type="Italic">Phoma</Emphasis> species,an endophytic fungus from the Chinese medicinal plant <Emphasis Type="Italic">Arisaema erubescens</Emphasis>

Through bioassay-guided fractionation, the EtOAc extract of a culture broth of the endophytic fungus Phoma species ZJWCF006 in Arisaema erubescens afforded a new α-tetralone derivative, (3S)-3,6,7-trihydroxy-α-tetralone (1), together with cercosporamide (2), β-sitosterol (3), and trichodermin (4). The structures of compounds were established on the basis of spectroscopic analyses. Compounds 1, 2, and 3 were obtained from Phoma species for the first time. Additionally, the compounds were subjected to bioactivity assays, including antimicrobial activity, against four plant pathogenic fungi (Fusarium oxysporium, Rhizoctonia solani, Colletotrichum gloeosporioides, and Magnaporthe oryzae) and two plant pathogenic bacteria (Xanthomonas campestris and Xanthomonas oryzae), as well as in vitro antitumor activities against HT-29, SMMC-772, MCF-7, HL-60, MGC80-3, and P388 cell lines. Compound 1 showed growth inhibition against F. oxysporium and R. solani with EC₅₀ values of 413.22 and 48.5 μg/mL, respectively. Additionally, compound 1 showed no cytotoxicity, whereas compound 2 exhibited cytotoxic activity against the six tumor cell lines tested, with IC₅₀ values of 9.3 ± 2.8, 27.87 ± 1.78, 48.79 ± 2.56, 37.57 ± 1.65, 27.83 ± 0.48, and 30.37 ± 0.28 μM, respectively. We conclude that endophytic Phoma are promising sources of natural bioactive and novel metabolites.