Eukaryotic cytochromes c contain a buried water molecule (Wat166) next to the heme that is associated through a network of hydrogen bonds to three invariant residues: tyrosine 67, asparagine 52, and threonine 78. Single-site mutations to two of these residues (Y67F, N52I, N52A) and the double-site mutation (Y67F/N52I) were introduced into Saccharomyces cerevisiae iso-1-cytochrome c to disrupt the hydrogen bonding network associated with Wat166. The N52I and Y67F/N52I mutations lead to a loss of Wat166 while N52A and Y67F modifications lead to the addition of a new water molecule ( Water in and around proteins is recognized as being important to protein structure, function and stability (4 -6). Surface and bound water molecules have been identified in protein structures using crystallographic methods and NMR spectroscopy. Eukaryotic cytochromes c, the paradigms of electron transfer proteins, are ideally suited for investigating the structural and functional purposes of water-protein interactions (7). Crystallographic studies performed on the oxidized and reduced states of both tuna and yeast iso-1-cytochrome c proteins have indicated that a conserved and internally bound water molecule (Wat166), 1 along with the surrounding hydrogen bond network are central to the structural transition of cytochrome c between oxidation states (8, 9). The water molecule is adjacent to the heme and the hydrogen bonding network which is composed of conserved residues Asn 52 , Tyr 67 , and Thr 78 , which are also hydrogen bonded in ferrocytochrome c to the Met 80 sulfur which is one of the two heme iron ligands. The oxidation-reduction or redox potential that determines the direction of electron flow between electron transfer proteins is dependent upon the heme ligands and the surrounding peptide (10). In addition, the functional properties of cytochrome c are dependent on the oxidation state of the protein, and knowledge of the energetics of protein stability with respect to oxidation state is central to our understanding of function (11). For example, the addition of an electron to ferricytochrome c results in modified functional properties including a significant increase in stability (12).Several studies using classical genetic procedures or site directed mutagenesis have shown that the hydrogen bond network and Wat166 modulate redox potential and the stability of the protein (13-20). The high resolution three dimensional structures of the reduced and oxidized states of yeast iso-1-cytochromes c carrying mutations at position 52 and/or 67 have been recently reported (1-3). When compared to the wild type protein structure, these mutants show significant changes in their hydrogen bonding networks adjacent to the heme as well as either the displacement of the conserved internally bound Wat166 or the addition of a second internally bound water molecule.Recent investigations have shown that depending on the amino acid substitutions, varying degrees of change in the free energy of unfolding are observed for the two redox states of cyto...

The oxidation-reduction potential of eukaryotic cytochromes c varies very little from species to species. We have introduced point mutations into isoform 1 of yeast cytochrome c (iso-1-cytochrome c) to selectively engineer a protein with a higher redox potential. Of the ten different mutant proteins generated for the present investigation Y67R, Y67K and W59H were found to be non-functional. Three other mutant proteins, L32M, L32T and T49K, were functional, but too unstable for biophysical studies. Mutant cytochromes c K79S, K79T, Y48H and Y48K were purified and characterized. The Y48K mutant is the only one that exhibits a significant increase of +117 mV in redox potential compared with the wild-type protein while still supporting oxidative phosphorylation in vivo. Low temperature difference spectroscopy confirmed the formation of the holoprotein, while adsorption and CD spectroscopy reveal perturbations in the structure of Y48K iso-1-cytochrome c.

The oxidation—reduction potential of eukaryotic cytochromes c varies very little from species to species. We have introduced point mutations into isoform 1 of yeast cytochrome c (iso-1-cytochrome c) to selectively engineer a protein with a higher redox potential. Of the ten different mutant proteins generated for the present investigation Y67R, Y67K and W59H were found to be non-functional. Three other mutant proteins, L32M, L32T and T49K, were functional, but too unstable for biophysical studies. Mutant cytochromes c K79S, K79T, Y48H and Y48K were purified and characterized. The Y48K mutant is the only one that exhibits a significant increase of +117mV in redox potential compared with the wild-type protein while still supporting oxidative phosphorylation invivo. Low temperature difference spectroscopy confirmed the formation of the holoprotein, while adsorption and CD spectroscopy reveal perturbations in the structure of Y48K iso-1-cytochrome c.

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