Identification of cysteine ligands in metalloproteins using optical and NMR spectroscopy: Cadmium‐substituted rubredoxin as a model [Cd(CysS)4]2‐ center

• Published in: Protein science 1993-10, Vol.2 (10), p.1756-1764
• Main Authors: Henehan, Colin J., Pountney, Dean L., Vašák, Milan, Zerbe, Oliver
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Binding Sites; Cadmium - chemistry; Cadmium - metabolism; cadmium substitution; cadmium‐substituted rubredoxin; cadmium‐thiolate charge‐transfer bands; Chemical Phenomena; Chemistry, Physical; Circular Dichroism; Cysteine - chemistry; Cysteine - metabolism; cysteine ligation; Desulfovibrio - chemistry; electronic spectroscopy; Magnetic Resonance Spectroscopy; Molecular Sequence Data; NMR; Rubredoxins - chemistry; Rubredoxins - metabolism; Zinc Fingers; zinc‐finger proteins
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1002/pro.5560021019

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)4]2‐ complex. The 113Cd NMR spectrum of Cd‐Rd contains a single 113Cd 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 (1H‐113Cd) multiple quantum coherence (HMQC) and total coherence spectroscopy (TOCSY)‐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 3J(1Hβ‐113Cd) 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 β ‐C β ‐S γ ‐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 Jørgensen, the lowest energy transition has been attributed to a CysS‐Cd(II) charge‐transfer excitation (245, 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‐n X n centers.