Effect of replacing a conserved proline residue on the function and stability of bovine adrenodoxin.

A proline residue in the C-terminal part of the polypeptide chain is highly conserved among many [2Fe-2S] ferredoxins. To investigate the requirement for proline at this position, we constructed steric (4-108W), charged (4-108K), polar (4-108S) and non-polar (4-108A) truncated mutants of adrenodoxin and studied them for biological function and stability. Although the variants were expressed in Escherichia coli with a significantly lower yield compared with wild-type adrenodoxin, successful incorporation of the iron-sulfur cluster suggested their proper folding. Similar absorption, CD and EPR spectra indicated that the cluster environment was not affected by the mutations. No evidence for an essential role of Pro108 in determining the redox potential of adrenodoxin or its interactions with the redox partners was found. However, replacement of this residue results in a dramatic decrease in the overall protein stability. The differences in the Gibbs energy of unfolding at 37 degrees C, delta[delta(d)G(37 degrees C)], are -5.0, -7.8, -10.1 and -10.7 kJ/mol for 4-108A, 4-108S, 4-108W and 4-108K mutants, respectively, compared with 4-108P as a control. We conclude that the principle function of Pro108 is to stabilize adrenodoxin threefold: (i) through limitation of the conformation of the polypeptide chain in this region, (ii) through a hydrogen bond to Arg14 and (iii) favorable hydrophobic contacts.