Human Endonuclease III (EndoIII), hNTH1, is an FeS containing enzyme which repairs oxidation damaged bases in DNA. We report here the first comparative biophysical study of full-length and an N-terminally truncated hNTH1, with a domain architecture homologous to bacterial EndoIII. Vibrational spectroscopy, spectroelectrochemistry and SAXS experiments reveal distinct properties of the two enzyme forms, and indicate that the N-terminal domain is important for DNA binding at the onset of damage recognition.