Low temperature 65Cu NMR spectroscopy of the Cu+ site in azurin.

(65)Cu central-transition NMR spectroscopy of the blue copper protein azurin in the reduced Cu(I) state, conducted at 18.8 T and 10 K, gave a strongly second order quadrupole perturbed spectrum, which yielded a (65)Cu quadrupole coupling constant of +/-71.2 +/- 1 MHz, corresponding to an electric field gradient of +/-1.49 atomic units at the copper site, and an asymmetry parameter of approximately 0.2. Quantum chemical calculations employing second order Møller-Plesset perturbation theory and large basis sets successfully reproduced these experimental results. Sensitivity and relaxation times were quite favorable, suggesting that NMR may be a useful probe of the electronic state of copper sites in proteins.

Residue ionization in LpxC directly observed by 67Zn NMR spectroscopy.

The pH dependence of the solid-state (67)Zn NMR lineshapes has been measured for both the wild type (WT) and the H265A mutant of Aquifex aeolicus LpxC, each in the absence of substrate (resting state). The (67)Zn NMR spectrum of WT LpxC at pH 6 (prepared at 0 degrees C) contains two overlapping quadrupole lineshapes with C q values of 10 and 12.9 MHz, while the spectrum measured for the sample prepared at a pH near 9 (at 0 degrees C) is dominated by the appearance of a third species with a C q of 14.3 MHz. These findings are consistent with the two p K a values previously observed by the bell-shaped dependence of the LpxC-catalyzed reaction. On the basis of comparison of the experimental results with predictions from quantum mechanical/molecular mechanical (QM/MM) modeling, we suggest that p K a1 (low pH) represents the ionization of Glu78 and p K a2 (high pH) reflects the ionization of another active site residue located near the zinc ion, such as His265. These results are also consistent with water being bound to the Zn (2+) ion throughout this pH range. The (67)Zn NMR spectra of the H265A mutant appear to be pH independent, with a C q of 9.55 MHz being sufficient to describe both low- and high-pH data. The QM/MM models of the H265A mutant suggest that over this pH range water is bound to the zinc ion while Glu78 is protonated.

A QM/MM approach to interpreting 67Zn solid-state NMR data in zinc proteins.

We present here a (67)Zn solid-state NMR investigation of Zn(2+) substituted rubredoxin. The sample has been prepared as both a dry powder and a frozen solution to determine the effects of static disorder on the NMR line shape. Low-temperature experiments have been performed at multiple fields to determine the relative contributions to the NMR line shape from the electric field gradient and the anisotropic shielding tensors. Finally we present the theoretical interpretation of the experimental results utilizing a combined quantum mechanical molecular mechanics (QM/MM) approach. Theory predicts a sizable contribution from anisotropic shielding as compared with previously examined model systems. This is in good agreement with the experimental data.

Zinc solid-state NMR spectroscopy of human carbonic anhydrase: implications for the enzymatic mechanism.

The pH dependence of the (67)Zn solid-state nuclear magnetic resonance spectroscopy of human carbonic anhydrase (CAII) has been investigated to characterize the nature of the fourth ligand. CAII, through the Zn(2+)-bound hydroxide, catalyzes the deceptively simple reaction: CO(2) + H(2)O <==> HCO(3)(-) + H(+). The accepted mechanism for CAII would predict that water would be bound to the Zn(2+) at pH 5 and hydroxide would be bound at pH 8.5. The measured values for the electric field gradient (EFG) or quadrupole coupling constant (Cq) for CAII are independent of pH within the limits of the experimental error, i.e., 9.8 +/- 0.2 MHz. The EFG interaction has been predicted by ab initio electronic structure calculations for water and hydroxide bound to the zinc, including various levels of hydrogen bonding. After comparing the predicted Cq's with the experimental values, we conclude that the species present from pH 5-8.5 is the hydroxide form. The NMR data presented here is not consistent with the accepted mechanism for CAII. We show that the NMR data is consistent with an alternative mechanism of CAII.

Low temperature solid-state NMR experiments of half-integer quadrupolar nuclides: caveats and data analysis.

Solid-state NMR spectroscopy of half-integer quadrupolar nuclides has received a lot of interest recently with the advent of new methodologies and higher magnetic fields. We present here the extension of our previous low temperature method to an 18.8T system. This new probe entailed a total redesign including a cross coil and variable capacitors that are operational at cryogenic temperatures. The limitations to sensitivity are also discussed; including a new diode network, the utilization of a cryogenic band pass filter, and the consequences of the RF profiles of the coil. Further, details of the spectroscopy of quadrupolar nuclei in a protein are discussed, such as the observation of the outer transitions and how to distinguish them from the desired +/-1/2 transition.