A recombinant DNA vector was generated by cloning the AXCP gene into the
pET26b(+) plasmid. Site directed mutagenesis was utilised to generate the desired mutants using the recombinant DNA plasmid as a template. The recombinant and all mutants were successfully expressed in the E. coli strain, BL2 l (DE3) and purified to not less than 95% using cation exchange chromatography.
At the haem binding site leucine in position 16 (L16) sits above the haem in the
distal site blocking direct access to the unoccupied sixth coordination site of the
haem. This residue is believed to a vital role in ligand binding and discrimination. To investigate the role of this residue, L16 was mutated to glycine (L16G), phenylalanine (L16F) and to tyrosine (L16Y). The side chain of tryptophan at position 56 (W56) blocks the solvent channel observed in type I cytochromes c 'which is the shortest route from the protein surface to the sixth coordinate position at the distal face of haem. This residue was mutated to glycine (W56G) to unblock the solvent channel and to tyrosine (W56Y) to investigate whether the side chain of tyrosine will stabilise the 6c adducts by providing H-bonding to the exogenous diatomic molecules upon their binding to the distal side of the haem. A double mutant; (L16F/W56G) was generated for further investigation of the effect of the channel.
Except for L16G, the ferric form of recombinant AXCP and all other mutants were
sensitive to change in the solvent pH. At alkaline pH, the spin state changed from the quantum-mechanically admixed high-spin/ intermediate-spin (S=⁵/_2, ³/ ₂) state to the pure high-spin (S=⁵/₂).
The spectroscopic studies of NO titration with the ferrous forms of proteins showed that the recombinant AXCP has formed a stable 5c-NO via the transient 6c-NO adduct. Except for L16G, all mutants have formed stable 5c-NO adducts but the transient 6c-NO adducts were undetectable in the timescale of the spectroscopic experiments. Among them, W56Y exhibited the highest reactivity towards NO.
Interestingly, L16G generated a stable 6c-NO adduct accompanied by an enhanced reactivity towards the NO. This is the first reported 6c-NO adduct for AXCP.
The ferrous forms of recombinant, L16F, L16Y and the double mutant L16F/W56G
were oxidised upon the CO titration and did not bind CO. This was attributed to the
decrease in the oxidation potential of these mutants upon the CO addition. Therefore, the excess concentration of reductant was kept in the reaction mixture to keep the oxidation potentials high enough preventing the oxidation. 1n this case all of them have formed a stable 6c-CO adducts. Other mutants; L16G, W56G and W56Y generated the 6c-CO adducts. These results show the effect of W56 and L16 on the oxidation potentials.
The crystal structures of recombinant AXCP at 1.15 A, L16G at 1.18 A, L 16Y at
0.98 A, and the double mutant L16F/W56G at 0.96 A resolution were determined.
The crystal structure of the recombinant AXCP exhibited a high degree of similarity to that of native protein. The overall structures of the mutants were similar to the recombinant AXCP. ln contrast to the crystal structures of recombinant and all the other mutants, L16G was already in the reduced form and 6c with histidine and a water molecule in the fifth and sixth coordination positions to haem. This is the first reported structure of AXCP that is 6c without the introduction of any exogenous ligands.