HETERONUCLEAR MAGNETIC-RESONANCE STUDIES OF ZN, CD-113, AND HG-199 SUBSTITUTED P-FURIOSUS RUBREDOXIN - IMPLICATIONS FOR BIOLOGICAL ELECTRON-TRANSFER

Nuclear magnetic resonance studies have been carried out on the iron-c ontaining protein rubredoxin from Pyrococcus furiosus, a marine hypert hermophilic archaebacterium that grows optimally at 100-degrees-C. Alt hough paramagnetic line broadening precluded NMR studies of the native , Fe+2,+3 protein, a new procedure for the substitution of iron by the divalent cations of the entire zinc triad enabled high resolution NMR studies of the zinc-, cadmium-, and mercury-containing proteins. Prev ious NMR and X-ray structural studies demonstrated that substitution o f iron by zinc does not alter the global protein folding or the struct ure of the metal binding site [Blake et al. Protein Science (1992) 1, 1522-1525]. In this paper, two-dimensional H-1 NMR spectroscopic analy sis reveals that substitution by Cd-113 or Hg-199 also does not lead t o a measurable structural perturbation. Two-dimensional H-1-Cd-113 and H-1-Hg-199 heteronuclear multiple quantum coherence (HMQC) and 1D spi n-echo difference NMR experiments have enabled the determination the i ntrinsic Cd-113 and Hg-199 chemical shifts and the identification of H -1-metal scalar coupled protons. This represents the first successful application of H-1-Hg-199 HMQC spectroscopy to a mercury-containing pr otein, and suggests that the method may be applicable to studies of Hg -containing proteins involved in bacterial mercury resistance such as MerR. The magnitudes of the Cys-Hbeta-Hg-199 and NH--S(Cys)-metal coup ling constants provide useful information regarding the structure of t he metal coordination site. Interestingly, protons of several hydropho bic residues that are in close proximity to the metal-coordinated cyst eine sulfur atoms exhibit scalar coupling to the metals. The coupling appears to occur via a so-called ''through space'' mechanism. Since sc alar coupling is actually mediated by electrons of overlapping orbital s, these findings have important implications for understanding the me chanism of intramolecular electron transfer in metallo-redox proteins, and specifically indicate that certain ''through-space'' electron jum ps may be energetically more favorable than previously expected.