Nanoscopic and redox characterization of engineered horse cytochrome C chemisorbed on a bare gold electrode.

In this paper, we exploit the potential offered by site-directed mutagenesis to achieve direct adsorption of horse cyt c on a bare gold electrode surface. To this issue, the side chain T102 has been replaced by a cysteine. T102 is close to the surface exposed C-terminal residue (E104), therefore the T102C mutation is expected to generate an exposed cysteine side chain able to facilitate protein binding to the electrode via the sulphur atom (analogously to what observed for yeast iso-1-cyt c). Scanning Tunnelling and Tapping Mode Atomic Force Microscopy measurements show that the T102C mutant stably adsorbs on an Au(111) surface and retains the morphological characteristics of the native form. Cyclic voltammetry reveals that the adsorbed variant is electroactive; however, the heterogeneous electron transfer with the electrode surface is slower than that observed for yeast iso-1-cyt c. We ascribe it to differences in the tertiary architecture of the two proteins, characterized by different flexibility and stability. In particular, the region where the N- and C-terminal helices get in contact (and where the mutation occurs) is analyzed in detail, since the interactions between these two helices are considered crucial for the stability of the overall protein fold.

Insights into the role of the histidines in the structure and stability of cytochrome c.

In this paper we investigate the role played by each histidine in the amino acid sequence of yeast iso-1-cytochrome c (with the exception of H18, the residue axially coordinated to the heme iron) in determining the protein structure and stability. To this end, we have generated and characterized the double mutants H26Y/H33Y, H26Y/H39K and H33Y/H39K obtained from the C102T variant of the protein, which retain only one histidine side chain in the amino acid sequence. In particular, the H39K mutation inserts a lysine at position 39 as in the sequence of equine cytochrome c. The H26Y/H33Y/H39K triple mutant, which lacks all three histidines, was also produced and its spectroscopic properties are compared with those of the double mutants. The data highlight the critical role played by H26 in determining protein stability. Recombinant horse cytochrome c and the corresponding H26Y mutant were also generated and characterized. Since equine cytochrome c exhibits higher stability than the yeast protein, this provides a valuable opportunity to understand the role played by the invariant H26 residue in determining structure and stability.

The 40s Omega-loop plays a critical role in the stability and the alkaline conformational transition of cytochrome c.

The structural and redox properties of a non-covalent complex reconstituted upon mixing two non-contiguous fragments of horse cytochrome c, the residues 1-38 heme-containing N-fragment with the residues 57-104 C-fragment, have been investigated. With respect to native cyt c, the complex lacks a segment of 18 residues, corresponding, in the native protein, to an omega (Omega)-loop region. The fragment complex shows compact structure, native-like alpha-helix content but a less rigid atomic packing and reduced stability with respect to the native protein. Structural heterogeneity is observed at pH 7.0, involving formation of an axially misligated low-spin species and consequent partial displacement of Met80 from the sixth coordination position of the heme-iron. Spectroscopic data suggest that a lysine (located in the Met80-containing loop, namely Lys72, Lys73, or Lys79) replaces the methionine residue. The residues 1-38/57-104 fragment complex shows an unusual biphasic alkaline titration characterized by a low (p K(a1)=6.72) and a high p K(a)-associated state transition (p K(a2)=8.56); this behavior differs from that of native cyt c, which shows a monophasic alkaline transition (p K(a)=8.9). The data indicate that the 40s Omega-loop plays an important role in the stability of cyt c and in ensuring a correct alkaline conformational transition of the protein.