Site-directed spin labeling (SDSL) in combination with EPR spectroscopy has proven to be a valuable tool to investigate structure and dynamics of proteins. Commonly, spin labels are introduced into proteins using cysteine residues. However, this strategy fails if proteins contain functionally important cysteines. A recent proof-of-principle study using T4 Lysozyme as a model system has shown that non-natural p-acetylphenylalanine binding the spin label through a ketoxime ligation can be used for SDSL [1]. While T4 Lysozyme does not contain essential amino acids and is a rather stable protein, the current contribution will present a study on sulfite oxidase, a molybdo-enzyme, which carries an essential cysteine residue binding the Moco-cofactor, using the same spin labeling strategy as in [1].Within this contribution we will discuss challenges involved in the labeling of a more fragile protein using the less reactive ketoxime ligation in comparison to the disulfide linkage and will explore the information gained from the EPR line shapes of these spin labels in terms of structure determination, using mutations along a helical turn as an exploratory example. In addition, doubly spin labeled proteins were created to determine distances between the spin labels using pulsed electron-electron double resonance (pELDOR/DEER) experiments. The distance distributions extracted from these measurements will be discussed in light of expectations based on simple geometric considerations and will be compared to results of cysteine based distance determinations.[1] M. R. Fleissner, E. M. Brustad, T. S. Kalai, C. Altenbach, D. Cascio, F. B. Peters, K. L. N. Hideg, S. Parker, et al., Site-directed spin labeling of a genetically encoded unnatural amino acid, Proc. Nat. Acad. Sci. 2009, 106, 21637.