Magnetic susceptibility studies of metalloproteins are potentially of great value. Few other techniques can measure the magnetic properties of integer spin states and of other paramagnetic states which are EPR-silent for reasons such as relaxation broadening. The difficulty with susceptibility studies of metalloproteins has been the lack of sensitivity of susceptometers leading to a requirement for highly concentrated samples (greater than 1000 MuM). The concentration procedure following optical or EPR characterization often causes changes in the sample's redox state. Ambiguity in the redox state of the suceptibility sample, uncertainties in the level of the sample's impurity paramagnetism, and the intrinsic lack of resolution of the static susceptibility method when compared with resonance techniques such as EPR and Mossbauer spectroscopy have led to unreliable interpretation of susceptibility experiments. Moreover, a fundamental problem for high molecular weight proteins has been the impossibility of achieving the concentration necessary for a susceptibility experiment. We have developed new techniques to measure the magnetic properties of low concentration (greater than 50 MuM) metalloprotein samples using a commercial superconducting SQUID susceptometer. We have combined this new capability with EPR and Mossbauer spectroscopy by developing a multi-instrument sample holder. We can now study the same frozen metalloprotein sample (without thawing the sample or changing its concentration or holder) with both EPR and Mossbauer prior to its study with the susceptometer. With this combination of techniques the redox state of the susceptibility sample can be fully defined and its level of impurity paramagnetism measured making reliable interpretation of the susceptibility data possible. In addition, new experiments combining photolysis at low temperatures with susceptibility measurements are now feasible. We will study bacterial and mammalian cytochrome oxidase, a bacterial Rieske protein and other representative [2Fe-2S] proteins, E. coli sulfite reductase, and D. gigas ferredoxin II. One of our goals is to lay the ground work for susceptibility studies of biochemical kinetics at room temperature.
