NMR of Paramagnetic Metalloproteins

BERTINI, I; TURANO, P; ALEJANDRO JOSE VILA

CHEMICAL REVIEWS.

AMER CHEMICAL SOC

Año: 1993 vol. 93 p. 2833 - 2932

0009-2665

Paramagnetic compounds are characterized by the presence of unpaired electrons. Compounds with unpaired electrons usually contain one or more transition metal ions. Radicals are also paramagnetic but they are not suitable for high-resolution NMR experimenta (as it happens also with some metal ions) for reasons which will be discussed later. Here we deal with NMR spectroscopy applied to metalloprokina containing one or more transition metal ions bearing unpaired electrons. We limit the interest to the highresolution experiments, i.e., to studies aimed at the assignment of the protein signals, sometimes in the presence of cofactors, substrates, and inhibitors. We will not discuss here the NMR investigation of nuclei belongingtosubstratesand inhibitors in rapidexchange between free and protein-bound forms, which is aimed at obtaining exchange parameters or at the mapping of such molecules within the enzymatic cavities. Analogously, we will not discuss the investigation of water molecules interacting with the metal ion in rapid exchange with the bulk water. Reviews dealing with water and other exogenous ligands interacting with paramagnetic proteins are available.14 The choice of discussing only the protein as a whole is due to a flourish of results in this area since the progress in NMR technology is forever yielding more refined methods for investigation. Nowadays a few research groups interested in this field attempt to do with paramagnetic macromolecules what has been done with the diamagnetic ones, i.e. solving the threedimensional (3D) structure in solution. One of the most relevant effects of the presence of unpaired electrons is a considerable line broadening of the NMR signals corresponding to nuclei in the neighborhood of the paramagnetic This is a very severe limitation for high-resolution NMR. However, other effects of the paramagnetism may be exploited in order to overcome this problem. Since the broadened signals are often outside the diamagnetic region of the spectrum and well spread among them, with especially adapted pulse sequences it is possible to detect their connectivities with other signals. If a broadened signal is under the diamagnetic envelope, it can still be located because its cross peaks in 2D experiments can be observed under particular experimental conditions.%l2 Under these conditions the cross peaks between the signals of slow relaxing nuclei have not developed to a level to be detected. With regard to the 1D NOE experiments, as they are carried out as difference s p e ~ t r a , ~on~lyJ ~si gnals which give NOES with the irradiated one are detected. This is another way to locate a broad signal under a complex envelope if it responds through NOE. The fast relaxing nature of nuclei sensing a paramagnetic center prevents us from performing all the experiments designed for slow relaxing nuclei but they are a challenge for NMR investigations in order to develop strategies aimed at the detection of either dipolar or scalar connectivities. A final point to be discussed here is related to the magnitude of the external magnetic field. At high magnetic fields, the separation between the electron Zeeman levels increases, and this induces a large electron magnetic moment. Upon molecular tumbling nuclear relaxation occurs. This effect is dramatic on the line width for large S and large magnetic fields (i.e. large electron magnetic moment) and for macromolecules with molecular weight larger than 30 000 (which experience large rotational correlation times). Therefore, whereas in general larger and larger magnetic fields are needed to increase resolution and sensitivity, in paramagnetic systems, eventually the intensity of the magnetic field can be a negative factor. The magnetic field that falls between 100 and 600 MHz can be the best compromise.