Rotational and translational water diffusion in the hemoglobin hydration shell: dielectric and proton nuclear relaxation measurements.

The dynamic properties of water in the hydration shell of hemoglobin have been studied by means of dielectric permittivity measurements and nuclear magnetic resonance spectroscopy. The temperature behavior of the complex permittivity of hemoglobin solutions has been measured at 3.02, 3.98, 8.59, and 10.80 GHz. At a temperature of 298 K the average rotational correlation time tau of water within a hydration shell of 0.5-nm thickness is determined from the activation parameters to be 68 +/- 10 ps, which is 8-fold the corresponding value of bulk water. Solvent proton magnetic relaxation induced by electron-nuclear dipole interaction between hemoglobin bound nitroxide spin labels and water protons is used to determine the translational diffusion coefficient D(T) of the hydration water. The temperature dependent relaxation behavior for Lamor frequencies between 3 and 90 MHz yields an average value D(298K) = (5 +/- 2) x 10(-10)m2 s-1, which is about one-fifth of the corresponding value of bulk water. The decrease of the water mobility in the hydration shell compared to the bulk is mainly due to an enhanced activation enthalpy.

Structural fluctuations and conformational entropy in proteins: entropy balance in an intramolecular reaction in methemoglobin.

The reversible intramolecular binding of the distal histidine side chain to the heme iron in methemoglobin is of special interest due to the very large negative reaction entropy which overcompensates the large reaction enthalpy. It may be considered as a prominent example of the ability of proteins (including enzymes) to provide global entropy in a local process. In this work new experiments and model calculations are reported which aim at finding the structural elements contributing to the reaction entropy. Geometrical studies prove the implication of the 20 residue E-helix being shifted by more than 2 A. Vibrational entropies are calculated by a procedure derived from the method of Karplus and Kushik. It turns out that neither the histidine alone nor the complete E-helix contribute more than 15 per cent of the required entropy.

A new method for the determination of the permittivity of small samples in the microwave range and its application to hemoglobin single crystals.

A cavity perturbation method for the absolute determination of the complex permittivity of small samples in the microwave range is developed and tested. Samples with volumes less than 0.4 mm3, for example protein powder or single crystals of macromolecules, may be investigated in a temperature range between 180 and 300 K, using this method. As an application the complex permittivity of hemoglobin single crystals is determined at three frequencies, v = 3.06 GHz, v = 8.76 GHz and v = 17.0 GHz in a temperature range between 180 and 300 K.

Conformational transition of aquomethemoglobin: intramolecular histidine E7 binding reaction to the heme iron in the temperature range between 220 K and 295 K as seen by EPR and temperature-jump measurements.

Temperature-dependent EPR and temperature-jump measurements have been carried out, in order to examine the high-spin to low-spin transition of aquomethemogobin (pH 6.0). Relaxation rates and equilibrium constants could be determined as a function of temperature. As a reaction mechanism for the high-spin to low-spin transition, the binding of N epsilon of His E7 to the heme iron had been proposed; the same mechanism had been suggested for the ms-effect, found in temperature-jump experiments on aquomethemoglobin. A comparison of the thermodynamic quantities, deduced form the measurements in this paper, gives evidence that indeed the same reaction is investigated in both cases. Our results and most of the findings of earlier studies on the spin-state transitions of aquomethemoglobin, using susceptibility, optical, or EPR measurements, can be explained by the transition of methemoglobin with H2O as ligand (with high-spin state at all temperatures) and methemoglobin with ligand N epsilon of His E7 (with a low-spin ground state). Thermal fluctuations of large amplitude have to be postulated for the reaction to take place, so this reaction may be understood as a probe for the study of protein dynamics.

A new method for absolute determination of radical concentrations of EPR.

A new method for precise absolute determination of radical concentrations by EPR is described. Cavity quality factors, cavity mismatching, power levels and receiver characteristics do not have to be measured. The measurement of radical concentration is reduced to the determination of geometrical factors, the area under the EPR absorption curve, the LF modulating field strength and a frequency change. Accuracies in the 0.5% range can be reached. The method is checked by and bared on complex function theory.

The synthesis of spin-label derivatives of NAD+ and its structural components and their binding to lactate dehydrogenase.

Spin-labelled derivatives of NAD+ and its structural components (i.e. adenosine, adenine, AMP, ADP and ADPR) have been synthesized. Their binding to pig heart lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) has been studied and dissociation constants have been determined. The spin-labelled derivatives of ADP and ADPR exhibit a tighter binding than the corresponding NAD+ derivative. This may be attributed to the repulsion of the positively charged nicotinamide ring by an histidine side chain in the active center of the enzyme.