SQUID measurement of metalloprotein magnetization. New methods applied to the nitrogenase proteins

Abstract

New techniques have been developed to exploit the sensitivity of a commercial SQUID susceptometer in the study of the magnetization of metalloproteins. Previous studies have ignored both the slow relaxation (hours) of spin I = 1/2 nuclei and residual ferromagnetic impurities in sample holders. These potential sources of noise were at or below the sensitivity of previous instruments. With these noise sources under control, one can now decrease the protein concentration by a factor of ten. In addition careful characterization of the frozen magnetization sample, including the use of a multi-instrument holder for combined study of the magnetization sample with Mössbauer spectroscopy, is required for reliable interpretation of the data in the face of paramagnetic impurities common to metalloprotein samples. Many previous magnetic studies of metalloproteins have been carried out in the Curie region. Saturation magnetization studies down to 1.8 K and up to 5 T can determine zero-field splitting parameters in addition to the spin and exchange coupling parameters measured in previous studies at lower fields and higher temperatures. Applications of these techniques to the study of the nitrogenase proteins of Azotobacter vinelandii are presented as examples.

Magnetic Characterization of Chromium Intermediates in the Reduction of Chromium (VI) by Glutathione in Acidic Solutions

“…All solutions were prepared in D 2 O to reduce the paramagnetic contribution of the proton nuclei of water whose nuclear paramagnetism (expressed as concentration of spins 1 2 accounts for about 0.26 mM). Because of this, the deuteron's contribution was only 0.02 mM [25]. A similar reason motivated the removal of oxygen from the samples.…”

Measurement of the spin concentration of metalloprotein samples from saturation-magnetization data with particular reference to cytochrome c oxidase

“…The sample space was modified to include a slow bleed of helium gas, controlled by a needle valve, which was introduced through the bottom of the sample support rod and passed out through the evacuation valve at the top of the sample tube. The data collection and processing methodology, together with a discussion of the requirement for deuteration of samples, has been reported elsewhere [3,4].…”

“…Unless stated explicitly to the contrary, fits reported herein are unique. Further details of the fitting procedure are described elsewhere [3,4]. The software used is now commercially available (WEB Research Co., Edina, MN, U.S.A.).…”

“…In such experiments, it is absolutely necessary to obtain an independent determination of the concentration of the paramagnet under investigation before information concerning its ground-state magnetic properties can be extracted. If, however, data is collected at multiple applied magnetic fields, over a sufficiently wide temperature range to include the saturationmagnetization region as previously suggested [3,4], then the spin concentration is determined by these data. In fact, it actually turns out to be undesirable to use an independently determined spin concentration in fitting saturation-magnetization measurements because a small error in the estimation of the amount of paramagnet present can lead to quite erroneous values being found for its spin Hamiltonian parameters.…”

“…Using recently developed methods of data collection and manipulation [3,4], the results of saturation-magnetization measurements on derivatives of cytochrome c, horseradish peroxidase and cytochrome c oxidase are reported. The particular samples were chosen to provide a series of data sets requiring a variable (increasing) number of free parameters in the spin Hamiltonian used to fit them successfully.…”