Hans Twilfer

4-Methoxybenzoate monooxygenase from Pseudomonas putida: Isolation, biochemical properties, substrate specificity, and reaction mechanisms of the enzyme components

Publisher Summary Studies on the mechanism of biological degradation of lignin or lignin model substances by fungi showed that degradation of lignin down to vanillic acid followed the pathway involving successively the intermediates: α-guaiacyl glycerolconiferyl ether, 4-hydroxy-3-methoxyphenylpyruvic acid, or 4-hydroxy-3-methoxycinnamic acid and vanillin. This chapter describes purification of the components of 4-methoxybenzoate monooxygenase. The enzyme activity of the cell-free crude extract is not proportional to protein concentration in the assay mixture, especially at low protein concentrations. This behavior shows that 4-methoxybenzoate monooxygenase is a dissociable enzyme system—that is, it consists of several components. This is confirmed by the isolation of two components, a reductase and a dioxygen-activating protein. The reconstitution of these two components reveals full enzymatic activity. The chapter also describes the properties of putidamonooxin (PMO). PMO, the dioxygen-activating component of the 4-methoxybenzoate monooxygenase, has a molecular weight of 126,000, as derived from ultracentrifugation and gel filtration.

Bohr-effect and pH-dependence of electron spin resonance spectra of a cobalt-substituted monomeric insect haemoglobin

The monomeric haemoglobin IV from Chironomus thummi thummi (CTT IV) exhibits an alkaline Bohr-effect and therefore it is an allosteric protein. By substitution of the haem iron for cobalt the O2 half-saturation pressure, measured at 25‡ C, increases 250-fold. The Bohr-effect is not affected by the replacement of the central atom. The parameters of the Bohr-effect of cobalt CTT IV for 25‡ C are: inflection point of the Bohr-effect curve at pH 7.1, number of Bohr protons -δlog p1/2 (O2)/gDpH=0.36 mol H+/mol O2 and amplitude of the Bohr-effect curve δlog p1/2 (O2)=0.84. The substitution of protoporphyrin for mesoporphyrin causes a 10 nm blue-shift of the visible absorption maxima in both, the native and the cobalt-substituted forms of CTT IV. Furthermore, the replacement of vinyl groups by ethyl groups at position 2 and 4 of the porphyrin system leads to an increase of O2 affinities at 25‡ C which follows the order: proto < meso < deutero for iron and cobalt CTT IV, respectively. Again, the Bohr-effect is not affected by the replacement of protoporphyrin for mesoporphyrin or deuteroporphyrin. The electron spin resonance (ESR) spectra of both, deoxy cobalt proto- and deoxy cobalt meso-CTT IV, are independent of pH. The stronger electron-withdrawing effect by protoporphyrin is reflected by the decrease of the cobalt hyperfine constants coinciding with g∥=2.035 and by the low-field shift of g∥. The ESR spectra of oxy cobalt proto- and oxy cobalt meso-CTT IV are dependent of pH. The cobalt hyperfine constants coinciding with g∥=2.078 increase during transition from low to high pH. The pH-induced ESR spectral changes correlate with the alkaline Bohr-effect. Therefore, the two O2 affinity states can be assigned to the low-pH and high-pH ESR spectral species. The low-pH form (low-affinity state) is characterized by a smaller, the high-pH form (high-affinity state) by a larger cobalt hyperfine constant in g∥. The correlation of the cobalt hyperfine constants of the oxy forms with the O2 affinities is discussed for several monomeric haemoglobins. The Co-O-O bond angle in cobalt oxy CTT IV is characterized by an ozonoid type of binding geometry and varies little during the pH-induced conformation transition. Due to the lack of the distal histidine in CTT IV no additional interaction via hydrogen-bonding with dioxygen is possible; this is reflected by the cobalt hyperfine constants.

Resolution enhancement of EPR spectra using the Fourier transform technique. Analysis of nitrosyl cytochrome c oxidase in frozen solution

Abstract A computer program is described which applies the Fourier transform technique to electron paramagnetic resonance spectroscopy, in order to improve the signalto-noise ratio and to differentiate or integrate the EPR spectra. Resolution enhancement is performed by transforming the lineshape from Lorentzian to Gaussian and by computing higher derivatives using the experimental first derivative spectrum. This procedure avoids overmodulation of the spectrum when higher derivatives are measured. Furthermore, a simulation procedure for spectra of randomly oriented anisotropic systems is combined with these Fourier transform facilities. As an example, the complete determination of the EPR parameters of nitrosyl cytochrome c oxidase complexes was accomplished. The hexacoordinated and the pentacoordinated complexes (without axially bound histidine) show hyperfine constants in the x and y directions (heme plane) which are equivalent for nitrosyl and pyrrole nitrogen atoms, respectively. Both complexes show a larger hyperfine splitting of 15 NO in the z direction than in the x and y directions. The interaction of the electron spin with the pyrrole nitrogens influences the resonances in the x and y directions significantly whereas the contribution to the resonances in the z direction seems to be very small. The hexacoordinated complex exhibits splitting constants of N ϵ of the histidine in all principal directions; the largest splitting is found in the z direction.

Conformation-controlled trans-effect of the proximal histidine in haemoglobins. An electron spin resonance study of monomeric nitrosyl-57Fe-haemoglobins.

A monomeric allosteric haemoglobin from Chironomus thummi thummi was reconstituted with 57Fe-haem. This reconstituted haemoglobin was found to be identical to the non-reconstituted material with regard to the O2-binding properties and the visible spectra. The 270 MHz proton magnetic resonance of the bis(cyano)-57Fe-haemin shows that the reconstituted haem is identical with the non-reconstituted haem. Furthermore it has been proved by proton magnetic resonance that in Chironomus haemoglobins the prosthetic group is proto-haem IX. The ESR spectrum of the native nitrosyl haemoglobin demonstrates rhombic symmetry of the haem iron (gxx = 2.086, gyy=1.981, gzz=2.005) and hyperfine structures at gyy (aNε = 1.35 mT) and at gzz (a15NO = 3.05 mT, a14NO = 2.19 mT, aNε=0.715mT, a57Fe = 0.38 mT). The spectrum is independent of pH and can be classified as a type II spectrum following the classification of ref. 2. NO-binding obviously stabilizes the tertiary structure of this haemoglobin in a “tense” conformation with a relatively strong o bond of the 5th ligand (Nε of imidazole) and a relatively weak o bond of the 6th ligand (NO). Reaction of this haemoglobin with anionic, cationic and non-ionic detergents, respectively, leads to a transformation of the NO-ligated form into a “relaxed” conformation with a stretched or broken a bond of the 5th ligand (Nε of imidazole) and a strong σ bond of the 6th ligand (NO). The ESR spectrum of this modified NO-haemoglobin shows again a rhombic symmetry of the haem iron (gxx = 2.10, gyy = 2.06, gzz=2.010), but dramatically changes in the g tensors (low field shift), hyperfine structures and hyperfine splitting constants (a15NO=2.32 mT, a14 NO = 1.66 mT, a57Fe = 0.48 mT). The hyperfine splitting is isotropic. Transition from the “tense” conformation to the “relaxed” conformation corresponds with an increase of the spin density at the iron atom by 26% and a decrease of the spin density at the NO ligand by 25%. The spin density at the Nε of imidazole strongly decreases in the “relaxed” conformation, so that a hyperfine splitting of this ligand is not any more resolved. These results demonstrate the trans-effect of the proximal imidazole which in haemoglobins controls the binding properties of the external ligand in trans-position.

Electron spin resonance spectra of penta- and hexacoordinated nitrosyl iron protoporphyrin IX complexes

Electron spin resonance (ESR) spectra of frozen aqueous solutions of NO · haem · base complexes and NO · haem intercalated into dodecyl sulfate micelles have been measured at 77 K and analyzed for the hyperfine components of 15NO,14N-base, 14N-pyrroles and 57Fe which coincide with the principal directions of the g tensor. The influence of the basicity of the nitrogen base on the spin distribution and geometry of the Fe-N-O grouping has been demonstrated by replacing imidazole for pyridine and by comparing the ESR spectra with those obtained for the monomeric insect haemoglobin CTT IV.The comparison of the hyperfine parameters described for the so-called pentacoordinated nitrosyl complex of CTT IV with those of the NO · haem intercalated into detergent micelles has furnished evidence that the ESR spectrum of this conformation state of haemoglobin has to be definitely assigned to a pentacoordinated nitrosyl complex.The azz values increase with the following orders: CTT IV (2.98 mT) < imidazole complex (3.04mT) < pyridine complex (3.15mT) for 15NO, and pyridine complex (0.59 mT) < imidazole complex (0.67 mT) < CTT IV (0.70 mT) for the 14N-base. This result is in conformity with an increase of the σ donor and the π acceptor strengths of the nitrogen base in trans-position to 15NO. The ayy and axx components of 15NO and the 14N-base are strongly nonequivalent in the nitrosyl haemoglobin CTT IV, and less nonequivalent in the NO · haem · pyridine complex, indicating bending of the Fe-N-O grouping. The hyperfine components of the axial ligands coinciding with the x and y component of the g tensor are nearly equal for the NO · haem · imidazole complex.

EPR spectra of structurally modified rooflike folded hemins: Axial ligand-induced rhombicity

~: R = CMe 3 Iron porphodimethenes ! are hemiDs that have a roof-like folded porphyrin core due to the e,y-addition of methyl groups [I]. The t-butyl groups in 2 impose a sterical hindrance to the axial ligands X, thus allowing the isolation of ~, a momonuclear hydroxoiron(III)complex [2]. The EPR spectra [3] give the following results: The largest rhombicity is caused by methoxide (~h, ~), followed by phenoxide ard-azide (!~, ~9). Lack of rhombicity in ~ or ~ is explained by the mobility of the OH proton or a special orientation of the PhO group due to sterical hindrance. The folding of the macrocycle does not cause rhombicity (la, 2a).

EPR spectra of penta- or hexacoordinate nitrosyl hemes with sterically modified porphyrin cores

~: m = CMe 3 Nitrosyliron(II)porphyrins show a tendency to exist as pentacoordinate species, e.g., the octaethylporphyrin derivative, Fe(OEP)NO (!9) [1], and the porphodimethene derivative Fe(OEPMeg)NO (2a) [2] have been describe~. The-rooflike folding of the perphodimethene core does not seem to have ailarge effect on the EPR spectrum [3]; both __la and ~ add pyridine(Py)trans to NO when the toluene solution contains Py. Replacement of the methyl groups in ~ by t-butyl groups allows the formation of 3a which does not add Py as its spectrum remains unaltered in {he presence of Py. This is due to direct or indirect sterical hindrance by the t-butyl groups.

Spectroscopic and functional characteristics of ferredoxin from clostridium pasteurianum after substitution of the inorganic sulfur by selenium and tellurium

The oxidized protein is ESR-silent as the respective native protein. The fu l ly reduced form shows at l iquid helium temperature an ESR spectrum (gmI.94) with nearly rhombic symmetry (g l = 2.099, g2 = 1.937, g~ = 1.884) 13] This spectrum co r res ponds to t ha t of the ha l f -~educed (S I / 2 ) na t i ve f e r r e d o x i n , whereas the f u l l y reduced f e r r e d o x i n shows a Spectrum of a S = I system i 4 ] . An a d d i t i o n a l resonance appears at g = 5.164. The temperature behav iour of t h i s s igna l is in c o n t r a s t to t ha t of the h i g h f i e l d resonance, i . e . i t can be measured at 77 K. This resonance is not observed in the o x i d i z e d s ta te and must t h e r e f o r e belong to the c l u s t e r . The i n t e r p r e t a t i o n of t h i s s igna l as a h a l f f i e l d resonance of a m a g n e t i c a l l y coupled t w o c l u s t e r system wi th S = I 13] is to be excluded because of the c l u s t e r s t o i c h i o m e t r y and of the f i e l d p o s i t i o n of t h i s resonance. I t might be poss ib le t ha t t h i s resonance belongs to a l o w l y i n g exc i t ed s ta te wi th S = 3/2.

Non-equivalence and inverse allosteric response of the alpha and beta chains in haemoglobins. An electron spin resonance study of NO-ligated Hb Kansas.

Abstract The electron spin resonance (ESR) spectra of 15NO- and 14NO-ligated Hb Kansas have been measured at 77 K in the range of pH 5 to 10. At low pH the ESR spectrum is the composite of a type I and a type II spectrum which changes to another composite of a type I and type II spectrum at high pH. For the definition of type I and type II spectra and the correlation of these types with two tertiary conformation states see Overkamp et al., Z. Naturforsch. 31 c , 524 [1976]. Both, the type I and the type II spectra observed at low and high pH respectively are different with regard to g-tensors and hyperfine-splitting constants. Therefore at intermediate pH values the ESR spectra of NO-Hb Kansas are the composites of four spectral components. The assignments of the four spectral components to the a and the β chains are arrived at from the comparison of the ESR spectra of the α2Mmet β2NO and of the α2MNO β2NO species of Hb M Iwate. α and β chains are both characterized by a pH-dependent spectral transition from a type I to a type II spectrum. The chains are non-equivalent with regard to both the type I and the type II spectra. The type I spectra assigned to the a and the β chains are characterized by g*zz = 2.0095 with a hyperfine splitting of a*zz (15NO) = 2.36 mT and gzz = 2.0085 with a hyperfine splitting of a*zz(15NO) = 2.41 mT respectively. The type II spectra assigned to the α and the β chains are characterized by g*′zz = 2.005 and a hyperfine splitting of a*′zz (15NO) = 3.07 mT and g′zz=2.005 and a hyperfine splitting of a′zz (15NO) = 3.31 mT. The change of the hyperfine splitting at gzz during the transition from type I to type II corresponds to an increase of the spin density at the NO by about 25% in both types of chains. Comparison of type I spectra of the NO-ligated α and β chains respectively demonstrates that the spin density at the NO is larger in the β chains than in the oc chains. The spectral types are correlated with functional states defined by the kinetics of NO-binding. Binding of inositol hexaphosphate has no influence on the ESR spectra in the whole range of pH as it is expected if NO-Hb Kansas is in the quaternary T structure.