MAGNETIZATION STUDIES OF METALLOPROTEINS

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Precise magnetization data on small volume (less than 200 mu L), low concentration (more than 100 mu M) metalloprotein samples can now be obtained using a commercial SQUID susceptometer and techniques we have developed. This data can be properly interpreted only if paramagnetic impurities and sample heterogeneities are also measured using a variety of spectroscopic techniques - preferably applied to the magnetization sample itself. Using this approach it is possible to measure the spin, spin concentration, zero-field splitting parameters, and exchange coupling of paramagnetic centers in metalloproteins. We will study selected samples of 57Fe enriched cytochrome oxidase from T. thermophilus after characterization by Mossbauer spectroscopy. We will select for sample homogeneity and purity. Magnetization data of these selected samples will be used to measure the strength of the exchange coupling at the a3:Cua3 site. The active site of E. Coli sulfite reductase (and of spinach nitrite reductase) consists of a four-iron cluster coupled to a siroheme. This coupled site can accommodate two electrons. We will determine the spin of the singly reduced state of this enzyme. The siroheme in this singly reduced state may be in an unusual intermediate-spin ferrous state. We will study oxidized and reduced uteroferrin and its phosphate complexes to determine the exchange coupling of its two-iron center in each of these states. The magnetization and Mossbauer spectra of the phosphate complex of 5?Fe enriched reduced uteroferrin will be studied to understand the lack of EPR signal of this parmagnetic state. The number of c-type hemes in hydroxylamine oxidoreductase (HAO) is variously estimated between 7 and 11 per monomer. We will use the new magnetization techniques to measure the total heme concentration to plus/minus 5%. This, in turn, will be used to establish accurate extinction coefficients for preparations of this enzyme. We have developed a multi-instrument, boron nitride sample holder for combined study of 57Fe enriched magnetization samples with Mossbauer spectroscopy. We will extend this multi-instrument strategy beyond iron containing proteins by developing an optical cuvette for combined study of magnetization samples with low temperature