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The crystal structure of lignin peroxidase (LiP) from the white rot fungus Phanerochaete chrysosporium was refined to an R-factor of 16.2 % utilizing synchrotron data in the resolution range from 10 to 1.7 A. The final model comprises all 343 amino acid residues, 370 water molecules, the heme, four carbohydrates, and two calcium ions. Lignin peroxidase shows the typical peroxidase fold and the heme has a close environment as found in other peroxidases. During refinement of the LiP model an unprecedented modification of an amino acid was recognized. The surface residue tryptophan 171 in LiP is stereospecifically hydroxylated at the Cbeta atom due to an autocatalytic process. We propose that during the catalytic cycle of LiP a transient radical at Trp171 occurs that is different from those previously assumed for this type of peroxidase. Recently, the existence of a second substrate-binding site centered at Trp171 has been reported, by us which is different from the "classical heme edge" site found in other peroxidases. Here, we report evidence for a radical formation at Trp171 using spin trapping, which supports the concept of Trp171 being a redox active amino acid and being involved in the oxidation of veratryl alcohol. On the basis of our current model, an electron pathway from Trp171 to the heme is envisaged, relevant for the oxidation of veratryl alcohol and possibly lignin. Beside the opening leading to the heme edge, which can accommodate small aromatic substrate molecules, a smaller channel giving access to the distal heme pocket was identified that is large enough for molecules such as hydrogen peroxide. Furthermore, it was found that in LiP the bond between the heme iron and the Nepsilon2 atom of the proximal histidine residue is significantly longer than in cytochrome c peroxidase (CcP). The weaker Fe-N bond in LiP renders the heme more electron deficient and destabilizes high oxidation states, which could explain the higher redox potential of LiP as compared to CcP., (Copyright 1999 Academic Press.)

The 1H nuclear magnetic resonance characteristics of met-cyano and met-aquo hemoglobin from the sheep bile duct parasite Dicrocoelium dendriticum have been compared to those of other monomeric hemoglobins and myoglobins. By varying temperature and pH, it was found that the studied material is a mixture of several isozymes differing slightly in their structural features around the heme cavity. The heme in-plane rhombic asymmetry, as indicated by the spread of the heme methyl hyperfine shifts, is intermediate between that of sperm whale myoglobin and leghemoglobin. The proximal histidine is present and its dynamic properties, as probed by the exchange of the ring NH with bulk solvent protons, point towards a cavity more stable than those of sperm whale myoglobin and leghemoglobin. In the met-cyano form, an exchangeable proton was detected close to the iron center that was tentatively assigned to an arginine residue located three amino acid residues closer to the C terminus than the proximal histidine. The transition from the met-aquo form to the met-hydroxy form occurring at pH 8.1 and previously detected by optical methods was observed. Furthermore, consideration of the mean heme methyl hyperfine shift average indicates that the iron remains six-co-ordinate down to below pH 4.5 irrespective of an acid-transition (pK approximately 5) in the protein. However, the presence of a "pseudo" six-co-ordinate (i.e. high-spin, in-plane, five-co-ordinate) iron at pH values below the acid-transition pK cannot be excluded on the basis of the presently available data. The pH dependence of several resonances in both the met-cyano and met-aquo forms of the protein reflect a pK value compatible with the titration of a heme propionate.

Reconstitution of liver fluke (Dicrocoelium dendriticum) apo-hemoglobin with hemins selectively deuterated at specific positions has permitted the assignment of several heme resonances in the proton nuclear magnetic resonance spectrum of the Met-aquo and Met-cyano forms of the holoprotein. It was established that in the Met-aquo form the meso protons resonate at positions characteristic of a six-co-ordinated in-plane iron. From this, we deduced that the Met-aquo species retains a bound water molecule at pH values as low as 4.5. The orientation of the proximal histidine imidazole ring with respect to the heme group in the cavity was determined through the identification of the heme methyl signals and the analysis of the hyperfine shift pattern in the Met-cyano hemoglobin proton nuclear magnetic resonance spectrum. Compared to sperm whale myoglobin, the heme appears to be rotated by 180 degrees about the alpha, gamma meso-axis. Protein isomers with the heme group in a reversed orientation were not detected, even shortly after reconstitution. In the Met-cyano form, the resonances most affected by the Bohr transition were shown to arise from the heme propionates.

The spectroscopic properties of Dicrocoelium dendriticum met-hemoglobin, investigated between pH 3.8 and 10.5, display two proton-induced transitions with apparent pK values of 8.1 and 4.7. The spectral changes, over the pH region 6.5 to 10.5, correspond to the high to low spin transition usually observed in ferric hemoproteins. The relaxation time (tau = 3.2 ms at the pK) associated with this transition is closely similar to that observed in Aplysia limacina met-myoglobin, but approximately 1000-fold slower than that of sperm whale met-myoglobin. The spectral changes associated with the more acid transition are in the opposite direction and have not been resolved by the temperature jump method. The rate constants for the reaction of azide with Dicrocoelium dendriticum met-hemoglobin were measured between pH 3.8 and 6.7 by the temperature jump method. Two kinetic schemes, both consistent with the pH dependence of the apparent rate constant for binding of azide, were identified. No objective way of discriminating between the two models is available. However, one of them is amenable to a physical interpretation based on a comparison with the structural, kinetic and spectral properties of other monomeric hemoproteins.

Structural features of the heme and the heme cavity of the monomeric hemoglobin (Hb) from the platyhelminth Dicrocoelium dendriticum were investigated by optical and proton nuclear magnetic resonance spectroscopy. Using nuclear Overhauser effects (NOEs) from resonances assigned previously through isotope labeling, most hyperfine-shifted resonances could be attributed to individual heme and protein protons in the cyano-metHb complex. It was observed that the heme 2-vinyl group is held in the trans orientation by nearby residues, whereas the 4-vinyl group exhibits an equilibrium between cis and trans orientations. NOE experiments in 1H2O allowed the identification of exchangeable protons belonging to the proximal histidine residue (F8) and to a distal residue. Detailed analysis of the NOE patterns obtained from the distal labile proton to non-labile protons and among these latter protons leads to the conclusion that a tyrosine side-chain occupies the distal site E7. Optical spectra of the alkaline-metHb also lead to this view, in that they are not typical of a hydroxy-metHb complex but instead resemble that of a hemin-phenolate or human mutant (M-type) Hb with a tyrosine residue linked to the iron atom. Further evidence for a distal tyrosine residue stems from the occurrence of an unusually stable transient ferrous Hb-cyanide complex, formed upon reduction of cyano-metHb to deoxy-Hb with dithionite. We suggest that the stability of this intermediate is due to a slow re-orientation of a large distal side-chain prior to cyanide dissociation. The sequence of the E-helix, known from the partially determined primary structure, was realigned to accommodate these findings. A frame-shift by one residue now positions a tyrosine at the distal site E7 instead of the originally proposed glycine residue.