Reduced and oxidized cytochrome c4 exhibit differences in dynamics

The temperature-dependent dynamics of the fully reduced and fully oxidized forms of Pseudomonas stutzeri cytochrome c(4) have been studied by Mossbauer spectroscopy. Prior to the dynamic analysis, an efficient labelling strategy has been developed for the expression of highly enriched Fe-57 recombinant cytochrome c(4). Subsequently, the protein has been purifed to apparent homogeneity. Mossbauer 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 Mossbauer spectroscopy, similarities and differences between cytochrome c(4), cytochrome c and HiPIP are discussed. The obtained results reveal that ( a) cytochrome c(4) 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 c(4) than in cytochrome c(4) ( c) the reduced cytochrome c(4) is the most flexible at low temperatures, and (d) protein specific dynamics are most distinct in the oxidized protein.