Key Role of Phenylalanine 20 in Cytochrome c₃:  Structure, Stability, and Function Studies

Abstract

Aromatic residues in c-type cytochromes might have an important function in the folding and/or electron transferring properties of the molecule. In the tetraheme cytochrome c3 (Mr 13 000) from Desulfovibrio vulgaris Hildenborough, Phe20, is located between heme 1 and heme 3 with its aromatic ring close and almost parallel to the ring plane of heme 1. We replaced this residue by a nonaromatic hydrophobe residue, leucine, and analyzed the effects in terms of functional, structural, and physicochemical properties. While the F20L replacement did not have any strong effects on the heme region stability, a decrease of the thermostability of the whole molecule was observed. In the same way, the four macroscopic redox potentials were affected by the mutation as well as the flexibility of the surface loop around heme 4. The F20L replacement itself and/or this structural modification might be responsible for the loss of the intermolecular cooperativity between F20L cytochrome c3 molecules.

EPR investigation of the role of B10 phenylalanine in neuroglobin — Evidence that B10Phe mediates structural changes in the heme region upon disulfide-bridge formation

“…7). Aromatic residues such as Phe residues have been shown to have an important function in the folding and/or electron transferring properties of proteins [37]. This is important in relation to the putative redox functions suggested for Ngb [10].…”

Implication for buried polar contacts and ion pairs in hyperthermostable enzymes

“…1, the ArAT Ph structure is more compact due to shortened loops (colored in green) compared to those observed in thermophilic ( Thermus thermophilus , PDB ID: 1bjw) and mesophilic ( E. coli , PDB ID: 1ars). According to the thermodynamic database of proteins and mutants (ProTherm; http://gibk26.bse.kyutech.ac.jp/jouhou/protherm/protherm.html) [32], the highest T m of an enzyme measured directly by DSC was 121.6 °C for cytochrome c 3 from Desulfovibrio vulgaris [33], although the T m value of Ph CutA1 from P. horikoshii was reported recently to be 150 °C [34] .…”

Solution structure of a mutant of the triheme cytochrome PpcA from Geobacter sulfurreducens sheds light on the role of the conserved aromatic residue F15

“…In the case of DvHc 3 , in which F20 was also replaced by leucine, it was shown that the macroscopic redox potentials were globally affected [38]. Moreover, it was proposed by the authors that the intramolecular electron exchange between heme III and heme I was drastically affected by the F20L replacement [38]. In the highly homologous tetraheme cytochrome DvMc 3 , the roles of aromatic residues in Table 2 Heme geometry for PpcAF15L (this work) and PpcA [20] cytochromes in solution.…”

“…The structural motif formed by F15 and hemes I and III is also conserved in the tetraheme cytochrome c 3 family, in which F20 occupies an equivalent position. There are biochemical studies reported for F20 mutants of D. vulgaris tetraheme cytochromes c 3 isolated from the strains Hildenborough (DvHc 3 ) and Miyazaki (DvMc 3 ) [38][39][40]. In the case of DvHc 3 , in which F20 was also replaced by leucine, it was shown that the macroscopic redox potentials were globally affected [38].…”

“…There are biochemical studies reported for F20 mutants of D. vulgaris tetraheme cytochromes c 3 isolated from the strains Hildenborough (DvHc 3 ) and Miyazaki (DvMc 3 ) [38][39][40]. In the case of DvHc 3 , in which F20 was also replaced by leucine, it was shown that the macroscopic redox potentials were globally affected [38]. Moreover, it was proposed by the authors that the intramolecular electron exchange between heme III and heme I was drastically affected by the F20L replacement [38].…”