Biomedical subjects

K Gersonde

Publications and source records attributed to K Gersonde.

At least 37 records · Page 2Linked to original sources

PubMed1988

A nuclear overhauser study of heme orientational isomerism in monomeric Chironomus hemoglobins.

The nuclear Overhauser effect (NOE) was used to investigate heme orientation and to obtain assignments for all resolved resonances in the 1H-NMR spectrum of met-cyano Chironomus thummi thummi monomeric hemoglobins III and IV (Hb III and Hb IV). The only non-heme resolved resonance was found to be from Phe-38 (CD1), and NOE dipolar connectivity between this resonance and the heme 5- and 8-methyls was used to establish the absolute orientation of the heme for each heme-insertion isomer present. The assignments of resonances and heme disorder permitted structural comparisons between the various components, including those due to a point mutation in Hb III. Finally, the characteristic differences of NOE patterns to amino-acid protons from substituents on heme pyrroles I and II formed the basis for assigning resonances and heme orientation relative to native Hb IV for deuterohemin-reconstituted Hb IV, for which there are no X-ray data available.

Animals↗

PubMed1988

Resonance Raman evidence for an unusually strong exogenous ligand-metal bond in a monomeric nitrosyl manganese hemoglobin.

Resonance Raman spectroscopy has been employed to determine the vibrational modes of monomeric nitrosyl manganese Chironomus thummi thummi hemoglobin (CTT IV). This insect hemoglobin has no distal histidine. By applying various isotope-labeled nitric oxides (14N16O, 15N16O, 14N18O), we have identified the Mn11-NO stretching model at 628 cm-1, the Mn11-N-O bending mode at 574 cm-1 and the N-O stretching mode at 1735 cm-1. The results suggest a strong Mn11-NO bond and a weak N-O bond. The vinyl group substitution does not influence the nu (Mn11-NO), delta (Mn11-N-O) and nu (N-O) vibrations. The Mn11-NO stretching frequency is insensitive to distal histidine interactions with NO, whereas the N-O stretching frequency is sensitive. Nitric oxide also binds to Met manganese CTT IV to form an Mn111. NO complex which undergoes a slow but complete autoreduction resulting in the Mn11.NO species. In manganese meso-IX CTT IV, the Mn111. NO Mn11. NO conversion alters the intensities of the porphyrin ring modes at 342, 360, 1587 and 1598 cm-1, but shifts the frequencies at 1504 and 1633 cm-1 (in Mn111.NO) to 1497 and 1630 cm-1 (in Mn11. NO), respectively. The unshifted marker line at 1378 cm-1 reflects the fact that the pi electron densities of the porphyrin ring are the same in the two complexes.

Animals↗

PubMed1988

Effect of cadmium ions on dioxygen affinity and polyphosphate activity of human red blood cells.

The effect of cadmium ions on the dioxygen affinity, the time-dependent depletion of intracellular polyphosphates, and the elongation of human red blood cells (RBC's) was examined. The incubation of RBC's in the presence of 1 mM Cd2+ at 37 degrees C for more than one hour results in a decrease of the p50 value by 2.5-3.0 mmHg in comparison to controls. The p50 of stripped (phosphate-free) hemoglobin is not affected by the presence of 1 mM Cd2+ (p50 = 4.8 mmHg at pH 7.2 and 37 degrees C). Experiments with RBC cryolysates demonstrate an apparently competitive effect of 2.3-bisphosphoglycerate (DPG) with cadmium ions on the dioxygen affinity. From 31P NMR spectra, 31P T1 relaxation, and 31P T2 relaxation behavior a more direct evidence for DPG-Cd2+ complexation is obtained. 31P NMR spectra of RBC cryolysates also indicate DPG-Cd2+ complexation. The hydrolysis of free polyphosphates in RBC's incubated at 37 degrees C as monitored by 31P NMR spectra can be noticed after a three-hour lag phase (constant polyphosphate level). This lag phase is lengthened from three hours to four hours in the presence of Cd2+ ions. RBC elongation, as a measure of deformability, decreases slightly upon incubation with 1 mM Cd2+.

Cadmium↗

PubMed1987

Protein-structural heterogeneity in a non-allosteric monomeric insect hemoglobin monitored by proton magnetic resonance spectroscopy.

Proton NMR has revealed two modes of structural heterogeneity in the monomeric hemoglobin I of Chironomus thummi thummi, CTT I; rotational disorder caused by a 180 degree rotation of the heme about the alpha, gamma-meso axis (primary heterogeneity), which varies for each preparation or reconstitution of this hemoglobin, and a 'silent' amino acid replacement [Thr/Ala exchange in position 98(FG4)] in the vicinity of the heme group, which is invariant under all experimental conditions. The heme rotational disorder (primary heterogeneity) can be removed by reconstitution of CTT I with the symmetrical protoheme III. The secondary splitting is not affected; the ratio of intensities of the two types of resonance remains constant. The 8-methyl and 3-methyl and one of the alpha-vinyl proton resonances for the major heme rotational component and the 5-methyl and 1-methyl and one of the alpha-vinyl proton resonances for the minor heme rotational component have been identified and assigned by reconstitution with deuterium-labeled heme. Decoupling experiments have been employed to assign vinyl beta protons in cis and trans position to the respective vinyl alpha protons. Hyperfine shifts for the heme protons exhibited no pH influence above pH 6, in accord with the lack of the alkaline Bohr effect. Below pH 6, pH effects are most strongly reflected by the 8-methyl and 5-methyl proton resonances possibly reflecting titration of the propionate groups.

Amino Acid Sequence↗

PubMed1987

The effect of cadmium ions on 2,3-bisphosphoglycerate in erythrocytes studied with 31P NMR.

The interaction of cadmium ions with human red blood cell (RBC) 2,3-bisphosphoglycerate (DPG) wasa studied by 31P NMR. 31P NMR spectra and 31P T1 and 31P T2 relaxation times give evidence for cadmium-2,3-bisphosphoglycerate complexation in aqueous solution. 31P NMR spectra indicate the occurrence of a similar complexation in RBC cryolysates. The lag phase (constant polyphosphate level) prior to DPG hydrolysis in incubated red blood cells is lengthened in the presence of cadmium.

2,3-Diphosphoglycerate↗

PubMed1987

Haem-rotational disorder in monomeric allosteric cyano-Met insect haemoglobins monitored by resonance Raman spectroscopy.

The haem-rotational disorder (insertion of haem into globin rotated about the alpha, gamma-meso axis by 180 degrees) has been investigated in the cyano-Met form of the monomeric allosteric insect haemoglobins, CTT III and CTT IV, by resonance Raman spectroscopy. The effect of haem disorder on the resonance Raman spectra has been observed in proto-IX, deutero-IX, and meso-IX CTTs. Most importantly, in the absence of overlapping vinyl vibrations, we have identified two Fe-C-N bending vibrations at 401 cm-1 and 422 cm-1 (pH 9.5) for 57Fe deutero-IX CTT IV ligated with 13C15N-, which are attributed to the two haem-rotational components. One Fe-C-N bending mode at 422 cm-1 shows a pH-induced shift to 424 cm-1 (pH 5.5) indicating the t----r conformational transition, whereas the other bending mode is pH-insensitive, representing a non-allosteric component. By replacing the unsymmetrical porphyrins with the "symmetrical" protoporphyrin-III we eliminate the haem disorder. Then, sharpening of the Fe-N epsilon(His) (at 313 cm-1) and Fe-CN (at 453 cm-1) stretching modes is observed and a single Fe-C-N bending mode (at 412 cm-1) appears. In cyano-Met proto-IX CTT III two vinyl bending vibrations at 412 cm-1 and 591 cm-1 assigned by deuteration of the vinyl groups also reflect the haem disorder. The 412 cm-1 vinyl vibration is intensity-enhanced via through-space coupling with one of the Fe-C-N bending modes (at 412 cm-1). In the cyano-Met form of proto-III CTT III this vinyl vibration is shifted to 430 cm-1 resulting in a dramatic drop in intensity. It is most likely that the specific vinyl-protein interaction at position 4 in one of the haem-rotational components is the origin of the coupling between the Fe-C-N and vinyl bending modes. The Fe-N epsilon(proximal His) and the Fe-CN stretching vibrations as well as the Fe-C-N bending vibration have been identified by 54Fe/57Fe and 13C15N/12C15N/13C14N/12C14N isotope exchange.

Animals↗

PubMed1987

Resonance Raman evidence for the mechanism of the allosteric control of O2-binding in a cobalt-substituted monomeric insect hemoglobin.

The substitution of iron for cobalt in the monomeric insect hemoglobin CTT (Chironomus thummi thummi) III does not alter the Bohr effect for O2-binding. The cobalt substitution in this hemoglobin allows us to identify not only the O-O and Co-O2 stretching mode but also the Co-O-O bending mode by resonance Raman spectroscopy. The assignments were made via 16O2/18O2 isotope exchange. The modes associated with the Co-O-O moiety are pH-dependent. These pH-induced changes of the resonance Raman spectra are correlated with the t = r conformation transition. At high pH (high-affinity state) two unperturbed O-O stretching modes are observed at 1,068 cm-1 (major component) and 1,093 cm-1 (minor component) for the 18O2 complex. These frequencies correspond to split modes at 1,107 cm-1 and 1,136 cm-1 and an unperturbed mode at approximately 1,153 cm-1 for the 16O2 complex. At low pH (low-affinity state) the minor component becomes the major component and vice versa. The Co-O2 stretching frequency varies for approximately 520 cm-1 (pH 5.5) to 537 cm-1 (pH 9.5) indicating a stronger (hence shorter) Co-O2 bond in the high-affinity state. On the other hand, the O-O bond is weakened upon the conversion of the low- to the high-affinity state. The Co-O-O bending mode changes from 390 cm-1 (pH 9.5) to 374 cm-1 (pH 5.5). In the deoxy form the resonance Raman spectra are essentially pH-insensitive except for a vinyl mode at 414 cm-1 (pH 5.5), which is shifted to 416 cm-1 (pH 5.5).

Allosteric Regulation↗

PubMed1987

Age-dependent variation of T1 and T2 relaxation times of adenocarcinoma in mice.

The T1 and T2 values of adenocarcinoma EO 771 inoculated into the hind leg of mice are characterized and correlated with the histopathologic state of the tumor. Growth-dependent changes (indicated by a T1 of 630-910 msec and a T2 of 68-185 msec) can be separated into four characteristic phases. The increase in relaxation times in the early phases (A and B) is due to an increasing amount of viable tumor tissue relative to normal muscle tissue. In the later phases (C and D), a decline of the relaxation parameters is observed that is parallel to an increase in the fraction of necrotic tissue. By multiexponential analysis, two relaxation components (indicated by and, respectively) for T1 and T2 and the corresponding fractions alpha 1 and alpha 2 can be observed for both tumor and surrounding muscle tissue. A tissue criterion ("magnetic resonance fingerprint") is defined by a combination of these multiple parameters. This criterion allows separation of not only muscle and tumor tissue but also viable (early state) and necrotic (late state) tumor tissue.

Adenocarcinoma↗

PubMed1986

Proton-magnetic-resonance investigation of the dynamics of the conformational transition in allosteric monomeric insect hemoglobins.

1H-NMR spectra of the monomeric insect (Chironomus thummi thummi) hemoglobins CTT III and CTT IV were investigated in the pH range 5-10 to gain insight into the dynamics of the tense to relaxed (t in equilibrium r) conformational transition in the deoxy (at 200 MHz) and cyano-met (at 360 MHz) form. These hemoglobins exhibit a pH-sensitive O2 affinity (Bohr effect) which is linked to the conformational transition. Both hemoglobins are comprised of two components which show heme rotational disorder due to a 180 degrees rotation of the heme group about the alpha,gamma-meso axis. The heme rotational components differ remarkably in their Bohr effects [Gersonde, K. et al. (1986) Eur. J. Biochem. 157, 393-404]. Several of the hyperfine-shifted heme proton resonances in these hemoglobin derivatives show pH-induced line-broadening which is largest at pK = 7.46 (for the heme rotational component with large Bohr effect) or pK = 7.06 (for the heme rotational component with small Bohr effect) determined from the plots of chemical shift versus pH. The line broadening at pK approximately equal to 7.5, shown for the heme rotational component of cyano-met CTT IV with the largest Bohr effect, decreases in the following order in parallel with the pH-induced shift change: 4-H beta-c (1.20 ppm) greater than 3-CH3 (0.80 ppm) approximately greater than 4H alpha (0.76 ppm) approximately greater than 4H beta-t (0.73 ppm) greater than 8-CH3 (0.35 ppm). Decrease in temperature at the pK value also leads to line-broadening. At 4 degrees C the hyperfine-shifted resonance attributed to 3-CH3 is split into two resonances assigned to the t (low-pH form) and r (high-pH form) conformation, respectively. This temperature dependence confirms the t in equilibrium r exchange process as the origin of the pH-induced line-broadening. The Bohr proton exchange rate at the allosteric site is orders of magnitude larger than the t in equilibrium r exchange rate. Therefore, the proton-linked t in equilibrium r transition appears as a first-order reaction. The rate constant ktr for the t in equilibrium r transition at the pK in different hemoglobin derivatives ranges over 0.2-7 ms-1. Surprisingly, ktr is identical for deoxy and cyano-met CTT IV.(ABSTRACT TRUNCATED AT 400 WORDS)

Allosteric Regulation↗

PubMed1986

The cobalt-nitrosyl stretching vibration as a sensitive resonance Raman probe for distal histidine-nitrosyl interaction in monomeric hemoglobins.

The Co-NO stretching vibration has been assigned in the resonance Raman spectra of various cobalt-substituted monomeric hemoglobins by employing isotope-labeling of nitrosyl (14N16O, 15N16O, 14N18O). Monomeric hemoglobins with a distal histidine (sperm whale myoglobin and leghemoglobin) exhibit this vibration at 573-575 cm-1, whereas hemoglobins without distal histidine (elephant myoglobin and insect hemoglobin from Chironomus thummi thummi, CTT III) show this vibration in the range of 553-558 cm-1. The Fe-NO stretching vibration which occurs in the range of 554-556 cm-1 does not reflect the distal histidine-ligand interaction. Therefore, the Co-NO moiety which is isoelectronic with the Fe-O2 moiety is a good monitor for distal effects on the exogenous ligand of hemoglobins, especially due to the fact that in hemoglobins with distal histidine the Fe-O2 stretching vibration (567-572 cm-1) is similar to the Co-NO stretching vibration.

Animals↗

PubMed1986

Resonance Raman investigation of CO-ligated monomeric insect hemoglobins. Direct evidence for reciprocal changes in iron-axial ligand bonds induced by allosteric transitions.

The resonance Raman spectra of the two affinity states of the CO-ligated monomeric insect hemoglobins, Chironomus thummi thummi (CTT) III ad IV, have been investigated. We have identified (via 54Fe/57Fe and 13C18O/12C16O isotope exchange) the Fe-N epsilon(His) stretching mode at approximately 317 cm-1. This stretching mode changes from 329 (pH 5.5) to 317 cm-1 (pH 9.5) reflecting the pH-induced t in equilibrium with r conformational transition. The Fe-CO stretching mode is also pH-sensitive changing from 483 (pH 5.2) to 485 cm-1 (pH 9.2) in 57Fe CTT III . 13C18O complex. However the C-O stretching mode is pH-insensitive. The nonallosteric monomeric insect hemoglobin CTT I does not exhibit a pH-dependence of these vibrational modes. pH-Induced effects were also observed for a vinyl bending mode at 379 cm-1 (pH 9.5) in CTT III deuterated at the beta-carbons of the vinyls in position 2 and 4. It shifts to 390 cm-1 at pH 5.5. The other vinyl vibration at 573 cm-1 exhibits intensity enhancement via through-space coupling with the Fe-C-O bending mode. Our resonance Raman data provide the first direct evidence that the trans-effect is operative as a trigger mechanism for ligand-binding in monomeric allosteric insect hemoglobins. In going from the low-affinity to the high-affinity state, the Fe-N epsilon(His) bond becomes weaker, whereas the Fe-CO bond becomes stronger.

Allosteric Regulation↗

PubMed1986

Mechanism of the control of dioxygen binding in a dimeric cobalt-substituted insect hemoglobin. Resonance Raman evidence for cobalt-axial-ligand bond changes.

Resonance Raman spectroscopy has been used to investigate the allosteric control mechanism for O2 binding in a cobalt-substituted dimeric insect hemoglobin (CTT II), which exhibits a large Bohr effect due to a pH-induced transition between two ligand affinity states. Substitution of cobalt for iron in CTT II does not modify the Bohr effect, but permits the resonance enhancement (hence the detection) of Raman lines corresponding to the vibrations of the axial ligand-cobalt bonds. Using 16O2/18O2 isotope substitution the O-O and Co-O2 stretching and the Co-O-O bending mode have been assigned to the two affinity states of this hemoglobin: v (O-O) changes from 1152 cm-1 (pH 5.5; t conformation) to about 1125 cm-1 (pH 9.5, r conformation), v (Co-O2) from 512 cm-1 (pH 5.5) to 537 cm-1 (pH 9.5) and delta (Co-O-O) from 378 cm-1 (pH 5.5) to 390 cm-1 (pH 9.5). The Co-N epsilon (His) stretching mode has also been detected changing from 313 cm-1 (pH 5.5) to 307 cm-1 (pH 9.5). For the first time, reciprocal behaviour between the Co-N epsilon and Co-O2 bonds and between the Co-O2 and the O-O bonds in an allosteric hemoglobin are demonstrated. Furthermore, the pH sensitivity of a vinyl bending mode in the range of 411-415 cm-1 has been investigated and shown also to reflect the t in equilibrium with r conformation transition.

Animals↗

PubMed1986

Bohr effect in monomeric insect haemoglobins controlled by O2 off-rate and modulated by haem-rotational disorder.

The monomeric insect (Chironomus thummi thummi) haemoglobins CTT III and CTT IV show an alkaline Bohr effect. The amplitude of the Bohr effect curve of CTT IV is about twice as large as that of CTT III. In particular, at low pH a time-dependent 'slow' decrease in p50 upon cyclic oxygenation/deoxygenation is observed which is larger if dithionite, instead of ascorbate, is the reducing agent. The decrease of p50 (increase in affinity) correlates with the ratio of haem-rotational components exhibiting an increase of the 'myoglobin-like' haem-rotational component with high O2 affinity and high stability of the globin-haem complex. The replacement of protohaem IX by mesohaem IX and deuterohaem IX, respectively, causes an increase in O2 affinity following the order: proto less than meso less than deutero CTT Hbs. The Bohr effect, however, seems not to be affected by these porphyrin side-group substitutions. The O2 affinity is modulated by steric effects due to the substituents in position 2 and 4 via variation of the protein-haem interactions which influence the O2 release. The replacement of iron by cobalt in proto and meso CTT IV leads to an increase of the p50 by two to three orders of magnitude. Neither central metal nor vinyl replacement affect the Bohr effect. The natural CTT Hbs III and IV analyzed for mono-componential kinetic systems exhibit pH-dependent O2 off-rate constants: 300 s-1 (at pH 5.6) and 125 s-1 (at pH 9.7) for CTT III, and 550 s-1 (at pH 5.4) and 100 s-1 (at pH 9.0) for CTT IV. Inflection points and amplitudes of the log koff/pH plots correspond to those obtained from the Bohr effect curves indicating again a larger Bohr effect for CTT IV than for CTT III. In contrast, the O2 on-rate constants are pH-independent (kon = 1.15-1.26 X 10(8) M-1 s-1). Thus, the Bohr effect is completely controlled by the off-rate constants. Analysis for bi-componential kinetic systems employing the eigenfunction expansion method clearly identifies two kinetic components for proto-IX and deutero-IX CTT Hbs which can be attributed to the two haem-rotational components x and y (x and y differ due to an 180 degree rotation of the haem group about the alpha,gamma-meso axis; y is the myoglobin-like haem-rotational component).(ABSTRACT TRUNCATED AT 400 WORDS)

Chironomidae↗

PubMed1986

Acid Bohr effect of a monomeric haemoglobin from Dicrocoelium dendriticum. Mechanism of the allosteric conformation transition.

The dioxygen affinity of Dicrocoelium dendriticum haemoglobin was determined as a function of pH with a thin-layer diffusion technique. From the oxygen dissociation and association curves Hill coefficients h equal 1 were obtained throughout. Ultracentrifugation studies prove this haemoglobin to be monomeric irrespective of pH and ligation state. Thus, Dicrocoelium haemoglobin is a non-cooperative monomer. It has the highest O2 affinity so far known for any monomeric haemoglobin: its half-saturation pressure, p50 value, ranges at 25 degrees C from 0.016 mm Hg to 0.15 mm Hg (2.13-20.0 Pa) dependent on pH. Dicrocoelium haemoglobin shows an acid Bohr effect only and as such it constitutes a new class of haemoglobins. Its log p50 versus pH plot (Bohr effect curve) is characterized by a large amplitude, delta log p50 = 0.96, and an inflection point (Bohr effect pK) at pH 5.0. A model for the acid Bohr effect of D. dendriticum haemoglobin is proposed. By generalization, both the alkaline and the acid Bohr effect in various monomeric haemoglobins may arise from a single Bohr group complex (salt bridge).

Allosteric Site↗

PubMed1986

Echo shape analysis and image size adjustment on the level of echoes: improvement of parameter-selective proton images.

Parameter-selective imaging for tissue characterization is performed at low magnetic fields (0.24-0.28 T) employing echo trains with a large number of echoes (less than or equal to 50), high-power pulses, and the projection reconstruction method. Preprocessing of the raw echoes is important for the quality of the echo images which are the bases for further evaluation and generation of parameter-selective images. Two new algorithms are implemented in the image preprocessing routine TOMIKON: Echo shape analysis and image size adjustment. Echo shape analysis allows the detection and the removal of spikes in the echo signals to avoid image artifacts. Image size adjustment is performed by expanding and contracting the time basis of the echoes resulting in a size normalization of the echo images, an indispensable condition for generating diffusion images.

Head↗

PubMed1986

In vitro proton T1 and T2 studies on rat liver: analysis of multiexponential relaxation processes.

At 37 degrees C and 20 MHz, the T1 and T2 proton relaxation processes in intact rat liver tissue are multiexponential functions which in the majority of cases were decomposed into a major (alpha* approximately 90%, T1* = 374 ms, T2* = 58 ms) and a minor (alpha** approximately 10%, T1** = 130 ms, T2* = 181 ms) component. Both, T1 and T2, are temperature-dependent with a temperature shift of delta T1 = 1.5 ms/degrees C and delta T2 = 0.5 ms/degrees C, respectively. Storage of liver tissue at 4 degrees C and 37 degrees C led to remarkable changes of the T1 and T2 values. For T2 these changes occurred after a shorter storage time than for T1, but they are more pronounced for T1. To avoid such influences the relaxation measurements were performed within one hour after excision of the tissue. Even at 4 degrees C, long-term storage (greater than 3 h) must be avoided. A method for the quantitative determination of the fat content in liver based on multiexponential analysis of the T1 relaxation process was evaluated employing mixtures of triolein with liver homogenate. Triolein is a two-component system with T1* = 144 ms (alpha* = 62%) and T1** = 355 ms (alpha** = 38%). Finally, liver-specific protocol conditions were defined for in vitro relaxation studies.

Animals↗

PubMed1985

Iron-histidine stretching vibration in the deoxy state of insect hemoglobins with different O2 affinities and Bohr effects.

Resonance Raman spectroscopy has been employed to detect the iron-proximal histidine stretching mode in deoxyhemoglobins from insect larvae of Chironomus thummi thummi (CTT). With the excitation of 413.1 nm, we observe a sharp and intense line in the 220-224 cm-1 region. The assignment of this line to the Fe-N epsilon (His) stretching mode was made on the basis of a 3-cm-1 shift upon 57Fe/54Fe isotope substitution. The Fe-N epsilon (His) vibration is used to monitor the possible changes in the Fe-N epsilon (His) bond strength (hence bone length) in the deoxy state of the monomeric (CTT I, III, and IV) and dimeric (CTT II) insect hemoglobins. As these hemoglobins differ in O2 affinity, off-rate and on-rate constants, and in the Bohr effect, they are excellent model systems for investigating the mechanism of protein control of the heme reactivity. Some of these hemoglobins (CTT III, IV, and II) are allosteric, exhibiting two interconvertible conformational states with high and low O2 affinity at high and low pH, respectively. The Fe-N epsilon (His) stretching frequency does not correlate with the O2 affinity, the on-rate and the off-rate constants for different hemoglobins, for different conformational states, and for modified hemoglobins with different heme peripheral groups. This vibrational mode is insensitive to deuteration of the heme vinyl groups. It is important to note that the Fe-N epsilon (His) bonds in the high pH (high-affinity) and the low pH (low-affinity) states are identical. This implies that the O2 molecule, prior to binding, "sees" identical binding sites. Thus, the difference in free energy changes upon O2 binding is manifested only in the oxy form.

Animals↗

PubMed1985

Dioxygen-activating iron center in putidamonooxin. Electron spin resonance investigation of the nitrosylated putidamonooxin.

The mononuclear non-haem iron center is the dioxygen-binding site of putidamonooxin which is the dioxygen-activating component of the 4-methoxybenzoate monooxygenase. Replacement of dioxygen by nitrosyl leads to the formation of a rather stable Fe3+ X NO- complex which is characterized by electron spin resonance (ESR) at g approximately equal to 4 and g approximately equal to 2. The ESR features can be composed by two spectral components which are characterized by different tetragonal distortions of the axial symmetry. Binding of 4-hydroxybenzoate, which is the product of the enzymatic reaction, leads to the formation of an ESR spectrum with pure axial symmetry. After binding of 4-methoxybenzoate, i.e. the physiological substrate of the monooxygenase, only one spectral component, i.e. that with a small tetragonal distortion, is observed. Binding of substrate analogues, like 4-aminobenzoate and 4-trifluoromethylbenzoate, leads to a spectral heterogeneity with variable amounts of the ESR component with a large tetragonal distortion. Benzoate induces an ESR spectrum with only that spectral component with large tetragonal distortion. The iron-depleted substrate-free form of the enzyme, ligated with NO, also shows ESR heterogeneity, i.e. both spectral components overlap, with 60% of the component with large tetragonal distortion. Binding of 4-methoxybenzoate leads to the occurrence of a pure spectrum, i.e. with small tetragonal distortion, whereas binding of benzoate leads to a pure spectrum with large tetragonal distortion. Thus, the structural heterogeneity is removed by binding of both the ligand (NO) and substrate. The Fe3+ X NO- complex is discussed as an analogue of the native oxy complex Fe3+ X O2-.

Binding Sites↗