Paramagnetic Proton NMR Methods Used in Studying the Hemeprotein Subunit of Escherichia coli Sulfite Reductase11This work was supported by grants from the NIH and VA. The Duke Magnetic Resonance Spectroscopy Center was established with grants from the NIH, NSF, and the North Carolina Biotechnology Center.

This chapter discusses paramagnetic proton nuclear magnetic resonance (NMR) methods used in studying the hemeprotein subunit of Escherichia coli sulfite reductase. NMR spectroscopy of solutions containing paramagnetic metalloproteins is a rapidly developing, specialized area in the larger field of protein NMR. In the presence of a paramagnetic prosthetic group, hyperfine-shifted resonances may be observed outside the normal diamagnetic spectral envelope, and can often be used as a probe of the protein's electronic and molecular structure near the active site. The chapter presents some of the principles and techniques necessary to successfully utilize paramagnetic NMR spectroscopy. Structural information about the active site can be obtained from the NMR spectra of paramagnetic metalloproteins, particularly in the case of coupled metal systems. Resonance assignments can be achieved by a variety of techniques, including isotopic substitution, ID-NOE measurements, 2D NOESY/COSY maps, variable temperature intercepts, differential dipolar relaxation, and comparison with model compounds. The size and magnitude of hyperfine-shifted resonances along with their temperature dependence and relaxation properties are also valuable in understanding the electronic and magnetic properties of paramagnetic proteins.