Cytochrome c' is a member of a unique family of class II cytochromes found in the periplasm of denitrifying, photosynthetic, methanotrophic, and sulfur-oxidizing bacteria. Structural investigations conducted so far show that all class II cytochromes are homodimer (apart from R. palustris cytochrome c', which is in solution as a monomer), with a penta-coordinate c-type haem per subunit covalently bound to the amino acid sequence CXXCH and a vacant sixth coordination site surrounded by hydrophobic residues. Cytochrome c' selectively binds NO and CO but not 0 2.
Spectroscopic data indicate that cytochromes c' share similarities with the
coordination chemistry and ligand binding properties of the soluble form of
guanylate cyclase (sGC), the mammalian receptor for nitric oxide which plays
important roles in cellular signalling. While no crystal structure for sGC has been
determined to date, crystallographic studies on Alcaligenes xylosoxidans cytochrome c' (AXCP) showed its ability to bind ligands to the Fe centre from both sides of the haem plane. In this context, cytochromes c' are proposed as the closest structural model in understanding the NO binding domain of sGC.
The spectral properties of ferric cytochromes c' are markedly dependent upon
solvent conditions such as the pH values and the polarity. An unusual spin state pH dependent was reported from EPR and electronic spectra of ferric cytochromes c', which was explained as a quantum-mechanically admixted high-spin/intermediatespin (S=5/2, 3/2) state (QS) and a pure high-spin (S=5/2) state (HS), with an increase of the latter at alkaline pH. The QS state arises when the energies of the pure high spin and intermediate spin states are very close, so that they can be coupled together.
Theoretical considerations attributed the presence of the QS state to a weak field
strength of the iron which could be weakened by either the hydrogen bonding of a water molecule to Na1 of the coordinated histidine, as appears in most of the
cytochromes c' structures, but not in all though-I removed it, and/or the presence of a positively charged residue, Lys or Arg in all cytochromes c', four residues after the His towards the C-terminal.
The establishment of a strong, rapid and economic expression system of cytochrome c' in E. coli is an important step in order to investigate the protein function and behaviour by spectroscopic and crystallographic methods as well as mutagenesis. Despite the use of plasmids carrying the ccm gene cluster encoding for c-type cytochromes maturation system, heterologous expression often results in low soluble product yield, apoprotein formation, or protein degradation. During this work an efficient expression system has been established for AXCP, resulting in a final yield ranging from 20 to 30 mg of protein/L of culture, with a purity of 2.8 (A4oo/ A280) .
This represents the highest expression level obtained among recombinant
cytochromes c'. In order to understand R124 role on cytochrome c' spin-state and NO binding, the positively charged residue was replaced by residues with hydrophobic (A and F), polar (Q), negative (E) and positive (K) side chains.
The distal pocket of the AXCP haem is occupied by two residues, L16 and F59,
which are proposed to be responsible for the discrimination of the ligand. This is
thought to occur because the side chains seem to mediate access to the distal face. Moreover, it has been proposed that such steric hindrance could be the cause of the prevalence of the penta-coordinate form of the nitrosyl-haem over the hexacoordinate one. In order to comprehend in more detail the haem accessibility, Leu16 was mutated to a smaller residue, such as alanine, and Phe59 to a tyrosine. The crystal structures of recombinant cytochrome c' from A. xylosoxidans together with the R124E and Rl24K cytochrome c' mutants have been determined. The mutant R124K structure solved at 0.98 A resolution represents the highest resolution structure of any cytochrome c' and, generally, in the haemoproteins. The considerable flexibility of the B-C loop and helices has been highlighted.
The crystal structures of recombinant cytochromes c' and its Rl24E and R124K
mutants are the first structures of recombinant A. xylosoxidans cytochrome c' and mutant proteins. The high structural similarity between the recombinant and native cytochrome c' protein support the results presented previously of their comparable spectroscopic characteristics.