FeS clusters containing proteins are structurally and functionally diverse and present in most organisms. Our understanding of FeS cluster production and insertion into polypeptides has benefited from collaborative efforts between in vitro and in vivo studies. The former allows a detailed description of FeS-containing protein and a deep understanding of the molecular mechanisms catalyzing FeS cluster assembly. The second allows to include metabolic and environmental constraints within the analysis of FeS homeostasis. The interplay and the cross talk between the two approaches have been a key strategy to reach a multileveled integrated understanding of FeS cluster homeostasis. In this chapter, we describe the genetic and biochemical/biophysical strategies that were used in the field of FeS cluster biogenesis, with the aim of providing the reader with a critical view of both approaches. In addition to the description of classic tricks and a series of recommendations, we will also discuss models as well as spectroscopic techniques useful to characterize FeS clusters such as UV-visible, Mössbauer, electronic paramagnetic resonance, resonance Raman, circular dichroism, and nuclear magnetic resonance.
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