Basic requirements for a metal-binding site in a protein: The influence of loop shortening on the cupredoxin azurin

The main active-site loop of the copper-binding protein azurin (a cupredoxin) has been shortened from C(112)TFPGH(117)SALM(121) to (CTPHPFM118)-T-112-P-115 (the native loop from the cupredoxin amicyanin) and also to (CTPHPM117)-T-112-P-115. The Cu(II) site structure is almost unaffected by shortening, as is that of the Cu(I) center at alkaline pH in the variant with the (CTPHPM117)-T-112-P-115 loop sequence. Subtle spectroscopic differences due to alterations in the spin density distribution at the Cu(II) site can be attributed mainly to changes in the hydrogen-bonding pattern. Electron transfer is almost unaffected by the introduction of the (CTPHPFM118)-T-112-P-115 loop, but removal of the Phe residue has a sizable effect on reactivity, probably because of diminished homodimer formation. At mildly acidic pH values, the His-115 ligand protonates and dissociates from the cuprous ion, an effect that has a dramatic influence on the reactivity of cupredoxins. These studies demonstrate that the amicyanin loop adopts a conformation identical to that found in the native protein when introduced into azurin, that a shorter than naturally occurring C-terminal active-site loop can support a functional T1 copper site, that CTPHPM is the minimal loop length required for binding this ubiquitous electron transfer center, and that the length and sequence of a metal-binding loop regulates a range of structural and functional features of the active site of a metalloprotein.