System the g value and its anisotropy and the a

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system, the g value (and its anisotropy) and the a values (hyperfine splitting from various nuclei and their anisotropy) are the major parameters reported. EPR spectroscopy has played a role in the development of Fe-S biochemistry akin to the role played by optical spectroscopy in the development of the biochemistry of the cytochromes,46-49 particu- larly for mitochondria 47 and chloroplasts, 50 where the g = 1.9 EPR signal has facilitated the monitoring of electron flow through these redox systems. Although EPR has been a powerful tool, it does have some important limitations. A necessary but not sufficient condition for EPR is that the center to be observed must be in a paramagnetic state. Fortunately, this condition is met for at least one member of each one-electron redox couple, i.e., the odd-electron species. However. even when the even-electron species is paramagnetic, it is usually not observed in the EPR, because of the presence of large zero-field splittings. Moreover, relax- ation effects and/or the population of excited states often cause the EPR of proteins to be unobservable at room temperature. necessitating the use of liquid N 2 or liquid He temperatures to observe the signals in the frozen state. The need to freeze samples prior to observation can lead to artifacts involving the observation of nonphysiological states and processes. On the positive side, the low temperature increases the signal intensity by altering the Boltzmann distribution of the spin population, and allows various quenching techniques to be used with EPR to evaluate kinetic and electrochemical parameters. Nevertheless, one cannot usually observe real-time kinetics or be certain that one is observing a physiologically relevant state. Despite these caveats, EPR has proven a valuable and, in some cases, indispensable tool for identification and monitoring of Fe-S sites. Recently, the advanced EPR techniques ENDOR (Electron Nuclear Double Resonance) and ESEEM (Electron Spin Echo Envelope Modulation) have allowed the extraction of additional information from the EPR signal. t Mossbauer spectroscopy measures nuclear absorption of light at y-ray energies, and can be used to probe nuclear energy levels (usually of 57Fe). The splitting of these levels is influenced by the (s) electron density at the nucleus, and by the electric-field gradient that is set up by nearby atoms. These factors affect the isomer shift and the quadrupole splitting of the Mossbauer spectrum, respectively. Information on nuclear hyperfine couplings is also available when experiments are conducted in the presence of an external (usually applied) magnetic field. Fortunately, the nucleus most commonly (and easily) studied by this technique is present in all the proteins discussed in this chapter, although the level of 57Fe (2 percent natural abundance) must be increased by isotopic enrichment to achieve a high-enough signal-to-noise ratio. For spectra containing one type of site, the spectra are relatively straightforward to interpret. For multisite systems deconvolution is
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