Haemoglobins have the ability to discriminate between oxygen and other diatomic molecules. To further understanding of this process the X-ray crystal structures of carbonmonoxy and nitrosyl-leghaemoglobin have been determined at 1.8 Å resolution. The ligand geometry is discussed in detail and the controversial issue of bentversuslinear carbon monoxide binding is addressed. The bond angle of 160° for CO-leghaemoglobin is in conflict with recent spectroscopy results on myoglobin but is consistent with angles obtained for myoglobin X-ray crystal structures. In contrast to the numerous carbon monoxide studies, very little stereochemical information is available for the nitric oxide adduct of haemoglobin. This is provided by the X-ray structure of NO-leghaemoglobin, which conforms to expected geometry with an Fe-NO angle of 147° and a lengthened iron-proximal histidine bond. Thus crystallographic evidence is given for the predicted weakening of this bond on the binding of nitric oxide.

The leghaemoglobins have oxygen affinities 11 to 24 times higher than that of sperm whale myoglobin, due mainly to higher rates of association. To find out why, we have determined the structures of deoxy- and oxy-leghaemoglobin II of the lupin at 1.7 A resolution. Results confirm the general features found in previous X-ray analyses of this protein. The unique feature that has now emerged is the rotational freedom of the proximal histidine. In deoxy-leghaemoglobin the imidazole oscillates between two alternative orientations, eclipsing either the lines N1-N3 or N2-N4 of the porphyrin; in oxy-leghaemoglobin it is fixed in a staggered orientation. The iron atom moves from a position 0.30 A from the plane of the pyrrole nitrogen atoms in deoxy- to a position in the plane in oxy-leghaemoglobin while the Fe- bond distance remains constant at 2.02 A. The Fe-O-O angle is 152 degrees, as in human haemoglobin. The oxygen is hydrogen-bonded to the distal histidine at N epsilon 2-O1 and N epsilon 2-O2 distance of 2.95 A and 2.68 A, respectively. The porphyrin is ruffled equally in deoxy- and oxy-leghaemoglobins, due to rotations of the pyrrols about the N-Fe-N bonds, causing the methine bridges to deviate by up to 0.32 A from the mean porphyrin plane. The only feature capable of accounting for the high on-rate of the reaction with oxygen are the mobilities of the proximal histidine and distal histidine residues in deoxy-leghaemoglobin. The eclipsed positions of the proximal histidine in deoxy-leghaemoglobin maximize steric hindrance with the porphyrin nitrogen atoms and minimize pi-->p electron donation, while its staggered position in oxy-leghaemoglobin reverses both these effects. Together with the oscillation of the imidazole between the two orientations, these two factors may reduce the activation energy for the reaction of leghaemoglobin with oxygen. The distal histidine is in a fixed position in the haem pocket in the crystal, but must be swinging in and out of the pocket at a high rate in solution to allow the oxygen to enter.

Complex of leghemoglobin with isoquinoline (Lb-IQ) is of interest with respect to the bulky ligand location in the heme pocket. The atomic model was refined (1.8 angstrom resolution) by a restrained parameter least-square procedure to a R factor of 0.18. The angle between the heme and ligand planes is 104-degrees, the non-coplanarity apparently being due to the heme pocket size. The atom nearest to Fe is one of C atoms (Fe-C distance is 1.7 angstrom) but not the N atom of the ligand as was supposed previously. The imidazole ring of the proximal His F11 residue is not positionally affected as compared with deoxy-Lb. In both complexes the Fe atom is displaced from the heme plane towards the proximal His residue, distance between Fe and the heme plane being 0.08 angstrom. The isoquinoline binding causes conformational changes, leading to some alterations in hydrogen bonds. The model includes 166 water molecules.

Circular dichroism (CD) spectra in the range of 350-650 nm were studied for a number of ferro- and ferriderivatives of lupin leghemoglobin: Lb(II), LbNO, LbO2, LbCO, LbNb and Lb(III).H2O, LbF, LbAc, LbCN, LbNc, LbIq respectively. It has been found that like soybean leghemoglobin and unlike myoglobin the majority of EC in Soret band has a negative sign, with the exception of LbCO and LbNb where due to the splitting of electron transitions polarized in the heme plane into x- and y-components positive EC appear. In 450-650 nm sometimes the splitting of Q0 and Q1 bands is observed. Changes in CD spectra are considered applying the data on geometry of octahedral complex of heme iron and conformational changes in the protein globule found during X-ray studies of the spatial structures of the complexes under consideration. CD spectra of nitrosobenzene- and oxyleghemoglobin were found to be the most different than the other ones. It is suggested that in the first case the peculiar pattern of the CD curve results from an additional aromatic group incorporated into heme surroundings, significant disturbance of the heme symmetry due to two ligand groups located over its plane and an open conformation of the heme pocket realized in this complex, in the second case - a unique for hemoglobins geometry of the O2 molecule bound as eta-2-ligand. It is shown that the CD spectra are most sensitive to the ligand nature when pi-orbitals of the ligand are included into interactions with iron d-orbitals.

A kinetic characterization was performed for inorganic pyrophosphatase from Thermus thermophilus. The optimum activity with Mg2+ as the activating metal ion lies in a pH range between 8.3 and 9.5. The hydrolysis of inorganic pyrophosphate is also activated by Zn2+, Mn2+, and Co2+. Calcium ions are not activating at all. Tripolyphosphate is another substrate hydrolyzed by the enzyme but only with Zn2+ as the activating metal ion. Other potential substrates like ATP and cyclic metaphosphates are not hydrolyzed even at high enzyme concentrations. Computer modelling of kinetic data obtained from activity measurements with different total magnesium ion and pyrophosphate concentrations confirms a kinetic model which was shown to be valid also for inorganic pyrophosphatases from other microbial sources. The corresponding parameter values are given. The inorganic pyrophosphatase from T. thermophilus exhibits extremely high thermostability which is decreased by addition of EDTA indicating a stabilizing effect of divalent metal ions.