Structural significance of an internal water molecule studied by site-directed mutagenesis of tyrosine-67 in rat cytochrome c.

Abstract

The tyrosine-67 to phenylalanine mutated rat cytochrome c is similar to the unmutated protein in its spectral, reduction potential, and enzymic electron-transfer properties. However, the loss of the 695-nm band, characteristic of the ferric form of the normal low-spin physiologically active configuration, occurs 1.2 pH units higher on the alkaline side and 0.7 pH unit lower on the acid side. Similarly, the heme ironmethionine-80 sulfur bond is more stable to temperature, with the midpoint ofthe transition being 300C higher, corresponding to an increase in AH of 5 kcal/mol (1 cal = 4.184 J), partially mitigated by an increase of 11 entropy units in AS. Urea has only slightly different effects on the two proteins. These phenomena are best explained by considering that the loss of one of the three hydrogen-bonding side chains, tyrosine-67, asparagine-52, and threonine-78, which hold an internal water molecule on the "left, lower front" side of the protein [Takano, Biol. 153,, is sufficient to prevent its inclusion in the mutant protein, leading to a more stable structure, and, as indicated by preliminary proton NMR two-dimensional phase-sensitive nuclear Overhauser effect spectroscopy analyses, a reorganization of this area. This hypothesis predicts that elimination ofthe hydrogenbonding ability of residue 52 or 78 would also result in cytochromes c having similar properties. It is not obvious why the space-filling structure involving the internalized water molecule that leads to a destabilization energy of about 3 kcal/mol should be subject to extreme evolutionary conservation, when a more stable and apparently fully functional structure is readily available.

Nitration of Solvent-exposed Tyrosine 74 on Cytochrome c Triggers Heme Iron-Methionine 80 Bond Disruption

“…3C). The effect of the Y67F replacement is in agreement with that reported for a similar mutation in rat cytochrome c, which was evaluated following the 695 nm band in the absorption spectrum (62).…”

“…Absorption and CD Spectroscopy of Nitrated Cytochrome c-The integrity of the sixth coordination position occupied by Met-80 in the native state of cytochrome c can be followed by inspecting the charge transfer band at 695 nm (62). The decrease or loss of the 695 nm band in mononitrated cytochrome c in either Tyr-97 or Tyr-74 at pH 7.4 suggested the possibility of an "early" alkaline transition (40) due to the decrease in pI (9.28 for native cytochrome c; (63)) determined by the addition of a -NO 2 group to tyrosine, known to decrease the pK a of the phenolic -OH group from ϳ10 to 7.5 (64).…”

Conversion of cytochrome c into a peroxidase: Inhibitory mechanisms and implication for neurodegenerative diseases

“…1C). Analysis of the electronic spectrum of Y67H mutant shows a shift by 2 nm of the absorption band at 695 nm that may be indicative of a slightly modified heme electronic structure, as already described for the mutation Y67F on rat cyt c [19]. As indicated by the amplitude of the 695 nm band, only a small effect, if any, is induced by mutation of Tyr67 on the Fe-S(Met80) bond integrity (Fig.…”

Effects of the Tyr64 Substitution on the Stability of Cytochrome c553, a low Oxidoreduction‐Potential Cytochrome from Desulfovibrio vulgaris Hildenborough

“…However, Tyr67 replacement by a phenylalanine residue in rat cytochrome c provides oxidoreduction properties and enzymic electron-transfer properties similar to those of the native protein, but an increase of pH 1.2 units for the pKalk and a decrease of pH 0.7 in the pKac were observed. These phenomena were explained by considering that the loss of one of the three hydrogen-bonding side chains Tyr67, Am52 and Thr78, which hold an internal water molecule, is sufficient to prevent the inclusion of the water molecule in the mutant protein leading to a more stable structure (Luntz et al, 1989). It is to be noticed that in the case of [Y64F]cytochrome c553, a slight increase of the pKalk and decrease of the pKac are observed.…”