IDENTIFICATION OF CYSTEINE LIGANDS IN METALLOPROTEINS USING OPTICAL AND NMR-SPECTROSCOPY - CADMIUM-SUBSTITUTED RUBREDOXIN AS A MODEL [CD(CYSS)4]2- CENTER

Optical and NMR methods are presented for the identification of cystei ne ligands in Cd-substituted metalloproteins, in particular those cont aining zinc-fingerlike motifs, using Cd-substituted Desulfovibrio giga s rubredoxin (Cd-Rd) as a model [Cd(CysS)4]2- complex. The Cd-113 NMR spectrum of Cd-Rd contains a single Cd-113 resonance with a chemical s hift position (723.6 ppm) consistent with tetrathiolate metal coordina tion. The proton chemical shifts of the four cysteine ligands were obt ained from one-dimensional heteronuclear (H-1-Cd-113) multiple quantum coherence (HMQC) and total coherence spectroscopy (TOCSY)-relayed HMQ C experiments. In addition, sequential assignments were made for two s hort cysteine-containing stretches of the polypeptide chain using a co mbination of homonuclear proton correlated spectroscopy, TOCSY, and nu clear Overhauser effect spectroscopy experiments, enabling sequence-sp ecific heteronuclear 3J(H-1(beta)-Cd-113) coupling constants for each cysteine to be determined. The magnitude of these couplings (0-38 Hz) follows a Karplus-like dependence with respect to the H(beta)-C(beta)- S(gamma)-Cd dihedral angles, inferred from the crystal structure of th e native protein. The difference absorption envelope (Cd-Rd vs. apo-Rd ) reveals three distinct transitions with Gaussian-resolved maxima loc ated at 213, 229, and 245 nm, which are paralleled by dichroic feature s in the corresponding difference CD and magnetic CD spectra. Based on the optical electronegativity theory of Jorgensen, the lowest energy transition has been attributed to a CysS-Cd(II) charge-transfer excita tion (epsilon245, 26,000 M-1 cm-1) with a molar extinction coefficient per cysteine of 6,500 M-1 cm-1. It is proposed that the intensity of this band can be used as a sensitive measure of the number of cysteine ligands present in Cd(CYSS)4-nXn centers.