Chapter 15 Isolation of Saccharomyces cerevisiae mitochondria for Mössbauer, EPR, and electronic absorption spectroscopic analyses.

Methods are presented to aid in the study of iron metabolism in isolated mitochondria. The "iron-ome" of mitochondria, including the type and concentration of all Fe-containing species in the organelle, is evaluated by integrating the results of four spectroscopic methods, including Mössbauer spectroscopy, electron paramagnetic resonance, electronic absorption spectroscopy, and inductively coupled plasma mass spectrometry. Although this systems biology approach only allows groups of Fe centers to be assessed, rather than individual species, it affords new and useful information. There are many considerations in executing this approach, and this chapter focuses on the practical methods that we have developed for this purpose. First, large quantities of mitochondria are required, and so published isolation methods must be scaled up. Second, mitochondria are isolated under strict anaerobic conditions to allow control of redox state and to protect O(2)-sensitive Fe-containing proteins from degradation. Third, the importance of packing mitochondria for both spectroscopic and analytical characterizations is developed. By measuring the volume of packed samples and the percentage of mitochondria contained within that volume, absolute Fe and protein concentrations within the organelle can be obtained. Packing samples into spectroscopy holders also affords maximal signal intensities, which are critical for these studies. Custom inserts designed for this purpose are described. Also described are the designs of a 25-L glass bioreactor, a mechanical cell homogenizer, a device for inserting short EPR tubes into the standard Oxford Instruments EPR cryostat, and a device for transferring samples from Mössbauer holders to EPR tubes while maintaining samples at liquid N(2) temperatures. A brief summary of what we have learned by use of these methods is included.