Reduced and oxidized cytochromec4 exhibit differences in dynamics

Abstract

The temperature-dependent dynamics of the fully reduced and fully oxidized forms of Pseudomonas stutzeri cytochrome c₄ have been studied by Mössbauer spectroscopy. Prior to the dynamic analysis, an efficient labelling strategy has been developed for the expression of highly enriched ⁵⁷Fe recombinant cytochrome c₄. Subsequently, the protein has been purified to apparent homogeneity. Mössbauer measurements were recorded in the temperature range 77–240 K for both protein forms. A detailed analysis of the high quality spectra is presented. Based on the information obtained from Mössbauer spectroscopy, similarities and differences between cytochrome c₄, cytochrome c and HiPIP are discussed. The obtained results reveal that (a) cytochrome c₄ exists in pure low spin electronic configuration in both oxidation states in the temperature range 77–240 K, (b) the heme pocket is more relaxed in cytochrome c₄ than in cytochrome c, (c) the reduced cytochrome c₄ is the most flexible at low temperatures, and (d) protein specific dynamics are most distinct in the oxidized protein.