Mutants azurin

Early NMRD studies on Cu(H)-azurin (Koenig and Brown, 1973), recently conftrmed and extended to a series of azurin mutants (Kroes... 

In order to probe the possible influence of aromatic residues on internal ET, an investigation was initiated on single-site azurin mutants in which Trp48 had been substituted by other amino acids, with both aromatic and nonaromatic side chains. In the experiments, the rate constants for intramolecular ET were determined as a function of temperature (23). The results are set out in Table I together with the standard free energies of reaction (AG°), the activation enthalpy (A// ) and activation entropy (AS ). It is clear that substitution of... 

The relevance of the Trp48 route is further confirmed by the pronounced deviation of two data sets from the linear relationship between the activation entropy and enthalpy (cf. Fig. 3), namely for WT A. faelcalis azurin ( ) and for the P. aeruginosa Val31Trp azurin mutant (V). The WT azurin from A. f is unique among azurins since it has Val at position 48 (instead of a Trp). The absence of the aromatic residue here is noteworthy, since Fig. 3 demonstrates that the point for this particular azurin clearly falls above the line (an unusually slow ET rate), whereas the point for the V31W mutant with two stacked aromatic residues falls below the line (reflecting the fast ET rate). 

In order to further distinguish between the possible pathways for ET in azurins, several azurin mutants were produced where two of the copper ligating amino acids, His46 and His 117, were systematically replaced by a noncoordinating glycine, while the third T1 ligand, Metl21, was replaced by a histidine... 

The reduction potential of P. aeruginosa azurin increases by 60-70 mV upon changing pH from 10 to 5 (38 0). The NMR studies addressed the question of how much protonation of the two conserved titratable histidines in azurin, His35 and His83, may increase the Cu(ll)/Cu(l). It was found that the contributions from these two residues are 50 and 13 mV, respectively (40). There is a problem here, however, since the reduction potential of the H35K azurin mutant displays essentially the same pH dependence as WT (39), which seems to speak against this hypothesis, unless the protonated lysyl E-amine group has an unusually low pAT value. 

Wild-type azurin including a single surface histidine at position 83 and Glnl07His/His83Gln azurin mutant were chemically modified by the luminescent rhenium(I) phenantroline complex 30. 

In other systems, W-band EPR and X-band Fe-ENDOR have been used to characterize a new stable diiron centre found in the E. coli RNR mutant R2-Y122H. The spectrum was fully resolved at 94 GHz and evidence was given to support the view that the centre was a Fe Fe " centre with a strongly coupled radical. Pulsed EPR at 95 GHz has been used to characterize the azurin mutant M121H, a blue copper protein, in a single-crystal study which allowed the complete g tensor to be determined relative to the molecular axes. ... 

Type 1.5 and type 2 copper-cysteinate proteins do not exist in nature they have all been constructed by site directed mutagenesis on different proteins, mainly azurin or Cu,Zn-superoxide dismutase. For the type 1.5 proteins, a recent crystal structure is available, the azide derivative of the Met 121-Ala azurin mutant. In addition, preliminary results on the Metl21-His azurin mutant, another type 1.5 protein, have also been presented. ... 

It is notable that Cu(NH3)3(SH)", which is a simple model of the type 1.5 Metl21-His azurin mutant has (p = 53-54°. Most probably, the spectral characteristics of the type 1.5 proteins are an effect both of the flattened tetragonal structure and the presence of a fourth strong ligand (in type 1.5 proteins the weak methionine ligand is replaced by a stronger ligand). 

---