Proton and metal ion-dependent assembly of a model diiron protein

Pasternak, A Kaplan, S Lear, JD DeGrado, WF

A. Pasternak et al., Proton and metal ion-dependent assembly of a model diiron protein, PROTEIN SCI, 10(5), 2001, pp. 958-969

44

INGLESE

Article

Biochemistry & Biophysics

PROTEIN SCIENCE

0961-8368 → ACNP

10

5

2001

958 - 969

ISI

0961-8368(200105)10:5<958:PAMIAO>2.0.ZU;2-#

DF1 is a small, idealized model for carboxylate-bridged diiron proteins. Th is protein was designed to form a dimeric four-helix bundle with a dimetal ion-binding site near the center of the structure, and its crystal structur e has confirmed that it adopts the intended conformation. However, the prot ein showed limited solubility in aqueous buffer, and access to its active s ite was blocked by two hydrophobic side chains. The sequence of DF1 has now been modified to provide a very soluble protein (DF2) that binds metal ion s in a rapid and reversible manner. Furthermore, the DF2 protein shows sign ificant ferroxidase activity, suggesting that its dimetal center is accessi ble to oxygen. The affinity of DF2 for various first-row divalent cations d eviates from the Irving-Willliams series, suggesting that its structure imp arts significant geometric preferences on the metal ion-binding site. Furth ermore, in the absence of metal ions, the protein folds into a dimer with c oncomitant binding of two protons. The uptake of two protons is expected if the structure of the ape-protein is similar to that of the crystal structu re of dizine DF1. Thus, this result suggests that the active site of DF2 is retained in the absence of metal ions.