Optical and NMR methods are presented for the identification of cysteine ligands in Cd-substituted metalloproteins, in particular those containing zinc-fingerlike motifs, using Cd-substituted Desulfovibrio gigas rubredoxin (Cd-Rd) as a model [Cd(CysS),]'-complex. The 'I3Cd NMR spectrum of Cd-Rd contains a single 'I3Cd resonance with a chemical shift position (723.6 ppm) consistent with tetrathiolate metal coordination. The proton chemical shifts of the four cysteine ligands were obtained from one-dimensional heteronuclear ('H-Il3Cd) multiple quantum coherence (HMQC) and total coherence spectroscopy (T0CSY)-relayed HMQC experiments. In addition, sequential assignments were made for two short cysteine-containing stretches of the polypeptide chain using a combination of homonuclear proton correlated spectroscopy, TOCSY, and nuclear Overhauser effect spectroscopy experiments, enabling sequence-specific heteronuclear J ( 'H8-"3Cd) 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 HP-CB-S7-Cd dihedral angles, inferred from the crystal structure of the native protein. The difference absorption envelope (Cd-Rd vs. apo-Rd) reveals three distinct transitions with Gaussian-resolved maxima located at 213, 229, and 245 nm, which are paralleled by dichroic features in the corresponding difference CD and magnetic CD spectra. Based on the optical electronegativity theory of Jmgensen, the lowest energy transition has been attributed to a CysS-Cd(I1) charge-transfer excitation 26,000 M-l cm-I) with a molar extinction coefficient per cysteine of 6,500 M-l cm-'. 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),+X, centers.

Far-UV CD, 'H-NMR, and Fourier transform infrared (FTIR) spectroscopy are three of the most commonly used methods for the determination of protein secondary structure composition. These methods are compared and evaluated as a means of establishing isostructural metal substitution in metalloproteins, using the crystallographically defined rubredoxin from Desulfovibrio gigas and its well-characterized cadmium derivative as a model system. It is concluded that analysis of the FTIR spectrum of the protein amide I resonance represents the most facile and generally applicable method of determining whether the overall structure of a metalloprotein has been altered upon metal reconstitution. This technique requires relatively little biological material (ca. 300 pg total protein) and, unlike either CD or 'H-NMR spectroscopy, is unaffected by the presence of different metal ions, thus allowing the direct comparison of FTIR spectra before and after metal substitution.Keywords: amide I band; far-UV CD; FTIR; isostructural metal replacement; metal substitution; NMR; rubredoxin; secondary structure In order to study the structure of a metalloprotein, it is often necessary to replace the native metal ion, for example Zn, by another metal, such as Co, ]I3Cd, or 57Fe, the properties of which enable the structure of the protein to be probed using a certain spectroscopic technique, such as UV-visible absorption, NMR, Mossbauer, etc. The methods used to investigate metalbinding sites in proteins have been reviewed recently (Coleman, 1993; Maret & Vallee, 1993; Munck et al., 1993). Although structural alterations may occur due either to the presence of a nonnative metal ion or to the preparative treatment used in obtaining a metalloderivative, the preservation of the native protein conformation in the metalloderivative is usually just assumed.The objective of the present work is to establish a suitable and facile spectroscopic method for confirming experimentally that the polypeptide fold of a metalloprotein is preserved following metal substitution. We have evaluated the suitability of three established methods for examining the secondary structure compositions of proteins that use CD, Fourier transform infrared (FTIR), and 'H NMR spectroscopic techniques, respectively, for comparing the structures of a native and metal-substituted "

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