Department of Chemistry and
Institute for Biomolecular Science
University of South Florida
Tampa, Florida 33620-5250
II. OVERVIEW OF NMR OF PARAMAGNETIC MOLECULES
A. Influence of Paramagnetism on NMR
B. NMR of Multinuclear Paramagnetic Metal Centers
III. COPPER,ZINC-SUPEROXIDE DISMUTASE
A. Cu,Co-Superoxide Dismutase
B. Cu,Ni-Superoxide Dismutase
C. Other Metal-Substituted Derivatives
IV. DINUCELAR IRON PROTEINS
A. Purple Acid Phosphatases
B. Hemerythrin and Other Dinuclear Iron-oxo Proteins
V. MULTINUCLEAR ZINC PROTEINS
B. Alkaline Phosphatase
C. Bacillus cereus Phosphotidylcholine-Specific Phospholipase C
VI. OTHER DI- AND MULTI-NUCLEAR METALLOPROTEINS
A. Iron-Sulfur Proteins
C. Cytochrome c Oxidase
A number of proteins have been characterized to contain a dinuclear or multinuclear metal center that are involved in many different kinds of biological processes and catalyses . These metal centers include the dinuclear Fe-oxo centers  in purple acid phosphatases , ribonucleotide reductase , methane monooxygenase , and fatty acid desaturase , the multinuclear Mn center in photosystem II , the multinuclear Cu center  in ascorbate oxidase, hemocyanine, tyrosinase, and the CuA site of cytochrome c oxidase , the Fe-S clusters in electron transfer ferredoxins , the Cu-Fe center in cytochrome oxidase , and the multinuclear Zn centers  in alkaline phosphatase, phospholipase C, nuclease P1, b-lactamase, and aminopeptidase. In most multinuclear metalloproteins, all the metal ions in the multinuclear metal center are acting cooperatively in catalysis, as observed in those dinuclear Fe-oxo proteins. However, in some other cases each individual metal has its own characteristic function and does not rely strictly on the status of the metal in the other sites, as observed in Cu,Zn superoxide dismutase. Thus, a better understanding of the action of multinuclear metalloenzymes relies on detailed studies of the mechanistic and structural roles each individual metal ion plays.
Paramagnetic transition metal-containing metalloproteins can be studied by the use of a number of different magnetic and spectroscopic methods owing to the presence of unpaired electrons on the metal ions. This is not the case for the large number of Zn-containing proteins. Nevertheless, the metal-binding site of Zn enzymes can be conveniently studied by the use of several paramagnetic metal ions as magnetic and spectroscopic probes for the Zn2+ [12,13], such as Cu2+ and Mn2+ as EPR probes  and Co2+ and Ni2+ as NMR probes . Studies of metalloproteins with spectroscopically active metal ions as intrinsic or external probes have provided further insight into the coordination chemistry of the active site in metalloproteins, including the coordination geometry, the coordinated amino acid side chains, the status of the coordinated water, and substrate and inhibitor bindings. The structural information can further our understanding of the reaction mechanisms of these metalloenzymes.
Metal substitution has been a well established method for the studies of metalloproteins in gaining information about their physical, chemical, and molecular properties and reaction mechanisms. This method becomes especially valuable for the investigation of spectroscopically silent Zn and Ca proteins. The rich optical and magnetic properties of transition metal ion probes allow us to study the metal binding sites in metalloproteins by the use of several different physical techniques. In many cases, the metal substituted derivatives retain high degrees of activity and can serve as good models for better understanding of the structural and mechanistic properties of the native enzymes, such as several Co2+-substituted Zn proteases [12,13]. In other cases, metal substitution may give rise to derivatives with "build-in inhibitors", the substituted metal ions, that can bind substrates tightly to form the ground-state E•S Michaelis complexes without proceeding with the subsequent catalysis (as opposed to the use of external inhibitors). An example can be found in the Co2+ and Cu2+ derivatives of the Fe2+ enzyme isopenicillin N synthase which form inert complexes with the tripeptide substrate d-(L-a-aminoadipoyl)-L-cysteiyl-D-valine. The study of these catalytically inert metal-substituted derivatives of metalloenzymes can provide insights into the structural and mechanistic properties of the transient enzymes-substrate complexes of the native enzymes.
Nuclear magnetic resonance (NMR) has been an indispensable tool for the study of molecular structure and dynamics, especially in biomolecular systems . In the past decade, NMR has also become a favorable tool of research in the study of metalloproteins, in establishing the identity of the endogenous ligands, probing the interaction of exogenous ligands with the active-site metal ions, and exploring the vicinity of the metal sites. Particularly, NMR studies of paramagnetic metalloproteins, metallo-biomolecules, and synthetic chemical models have become a new advanture [15,18] In this review, we discuss the studies of several dinuclear metalloproteins by the use of 1D and 2D NMR techniques that have been accomplished in the past few years. Three families of such proteins will be discussed in detail: Cu,Zn-superoxide dismutase and its metal-substituted derivatives, dinuclear m-oxo Fe proteins, and multinuclear hydrolytic Zn proteins. A brief account is also given on a few other multinuclear metalloproteins that have been investigated with NMR techniques.
(Back to Current Publications)
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