Cloning, expression and physicochemical characterization of a di-heme cytochromec4 from the psychrophilic bacteriumPseudoalteromonas haloplanktisTAC 125

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The 20-kDa di-heme cytochrome c4 from the psycrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 was cloned and expressed in Escherichia coli and investigated through UV-vis and 1H NMR spectroscopies and protein voltammetry. The model structure was computed using the X-ray structure of Pseudomonas stutzeri cytochrome c4 as a template. The protein shows unprecedented properties within the cytochrome c4 family, including (1) an almost nonpolar surface charge distribution, (2) the absence of high-spin heme Fe(III) states, indicative of a thermodynamically stable and kinetically inert axial heme His,Met coordination, and (3) identical E°′ values for the two heme centers (+0.322 V vs the standard hydrogen elecrode). At pH extremes, both heme groups undergo the “acid” and “alkaline” conformational transitions typical of class I cytochromes c, involving ligand-exchange equilibria, whereas at intermediate pH values their electronic properties are sensitive to several residue ionizations.

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