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Y64 has been replaced in cytochrome c553 from Desulfovibrio vulgaris Hildenborough by phenylalanine, leucine, valine, serine and alanine residues. An NMR study of structural variation induced in both oxidoreduction states of the molecule has been carried out by analysing observed chemical-shift variations. Dynamic changes were evidenced using NH exchange. We have observed that the substitution has a drastic effect on the stability of the molecule in the reduced state, although there is no effect on the reduction potential of the cytochrome. Y64-->F substitution induces particular effects on the NH exchange at the N-terminal, C-terminal and central alpha-helices and increases the stability of the oxidized molecule.

In order to study the conformational stability induced by the replacement of Tyr-64 in Desulfovibrio vulgaris Hildenborough (DvH) cytochrome c553, fast peptic digestion of deuterated protein followed by separation and measurement of related peptides using liquid chromatography coupled to electrospray ionization mass spectrometry was performed. We show that the H-bonding and/or solvent accessibility properties were modified by the single-site mutation. The mutant proteins can be classified into two groups: the Y64F and Y64L mutants with nearly unchanged deuterium incorporation compared to the wild-type protein and the Y64S, Y64V and Y64A mutants with increased deuterium incorporation. The 70-74 peptide was the most affected by mutation of Tyr-64, the phenylalanine mutant inducing slight stabilization whereas the serine mutant was significantly destabilized. In addition, from the analysis of the overlapping 37-57 and 38-57 peptides we can conclude that the amide proton of Tyr-38 has been replaced by deuterium in all proteins.

Hydrogen/deuterium exchange as well as charge state distribution monitored by electrospray ionization mass spectrometry were demonstrated to be a powerful and effective new tool for probing conformational properties of proteins in solution. In this paper, the influence of single amino acid replacements on the global conformation of cytochrome C553 from Desulfovibrio vulgaris Hildenborough using isotopic exchange monitored by electrospray ionization mass spectrometry is reported. Based on their respective charge state distributions and isotopic exchanges, we have differentiated relative stability of mutants and a ladder classification with the order being wild-type > Y64F = Y64L > Y64V > Y64A, under specific conditions of pH, is proposed.

Cytochrome c553 from sulfate-reducing bacteria is a low-oxidoreduction-potential cytochrome. The primary and tertiary structures show notable differences when compared to mitochondrial cytochromes. Tyr64 replacement in cytochrome c553 provides evidence that this residue is not directly involved in the potential modulation but is mostly implicated in the hydrogen-bond network around the heme. While the different variants obtained did not induce drastic structural modifications, they did affect the stability of the protein. This decrease of stability in acidic and alkaline environments was observed by variations in the optical spectra and by mass spectrometry. In addition, the mobility of aromatic side-chain was found to be increased in the mutant proteins as monitored by two-dimensional NMR spectroscopy.