Zhi Hao Zhou

Quantitative measurement of small through-hydrogen-bond and ‘through-space’1H-113Cd and1H-199Hg J couplings in metal-substituted rubredoxin fromPyrococcus furiosus

SummaryA method is described for measurement of small unresolvable heteronuclear J couplings. The method is based on quantitative analysis of a phase-purged heteronuclear spin-echo difference spectrum, and is demonstrated for measuring1H-113Cd and1H-199Hg J couplings in metal-substituted rubredoxin (Mr ∼ 5.4 kDa) fromPyrococcus furiosus. Couplings from cadmium to backbone amide protons that are hydrogen bonded to the Cys-S atoms directly bonded to Cd vary from smaller than 0.3 to 1.8 Hz; a ‘through-space’ coupling between Cd and the protons of an alanine methyl group was measured to be 0.3 Hz. Couplings to199Hg are significantly larger and fall in the 0.4–4 Hz range.

1H NMR investigation of the secondary structure, tertiary contacts and cluster environment of the four-iron ferredoxin from the hyperthermophilic archaeon Thermococcus litoralis

SummaryThe solution molecular structure of the four-iron ferredoxin (Fd) from the hyperthermophilic archaeon Thermococcus litoralis (Tl) has been investigated by 1H NMR spectroscopy. TOCSY and NOESY experiments in H2O, tailored to detect both weakly and strongly relaxed resonances, together with steady-state NOEs in both H2O and D2O, allowed the identification of 58 of the 59 residues, with one residue near the paramagnetic center undetected. It is shown that the contact shifted and strongly relaxed signals for all four cysteines ligated to the paramagnetic cluster can be assigned by standard backbone connectivities that do not require any assumptions about the tertiary structure. Secondary structural elements identified in Tl Fd are a three-stranded antiparallel β-strand involving the termini of the protein, a double β-strand (also antiparallel), two α-helices and four turns. The existence of a disulfide bridge between the nonligated cysteines is also proposed. Dipolar contacts observed in the NOESY maps and by steady-state NOEs between the ligated cysteines and the ‘diamagnetic’ protein matrix indicate that the overall folding pattern of Tl Fd is very similar to that of the 3Fe ferredoxin from the mesophilic bacterium Desulfovibrio gigas [Kissinger et al. (1991) J. Mol. Biol., 219, 693–723]. The influence of the paramagnetism of the cluster on the relaxation properties of the proton signals of nonligated residues near the cluster, as well as on the ligated cysteines, correlates well with the proximity to the cluster iron(s), as predicted from the crystal structures for homologous protons of other single-cluster ferredoxins. Finally, the potential role of the various identified structural factors in contributing to the hyperthermostability of this protein is discussed.

Two-dimensional gel electrophoresis mapping of proteins isolated from the hyperthermophile Pyrococcus furiosus

Abstract Two-dimensional gel electrophoresis (2DE) in polyacrylamide was used to map the proteins in lysates of the archaeon (formerly archaebacterium) Pyrococcus furiosus and to analyze enzymes purified from P. furiosus. The location of the enzymes in the 2DE maps was determined by comigration of lysate proteins with purified enzymes. A 2DE map of P. furiosus proteins with some identifications was produced, which will be useful for future studies of protein expression in this organism. In addition, the usefulness of 2DE for evaluating the purity of enzyme preparations and for characterizing their subunit structure under denaturing conditions was investigated.

X-ray absorption spectroscopy of Pyrococcus furiosus rubredoxin

Abstract X-ray absorption spectroscopy has been used to probe the frozen solution structure of the metal site in Pyrococcus furiosus rubredoxin in the native, iron-containing protein and in zinc- and mercury-substituted proteins. For all samples studied, the spectra have been interpreted in terms of a single shell of coordinated sulfur, with approximately tetrahedral coordination. For the native protein we obtain Fe-S bond-lengths of 2.29 and 2.33 Å for oxidized and reduced proteins, respectively. These values are in excellent agreement with those previously obtained from X-ray crystallography. The metal-substituted rubredoxins possess metal-sulfur bond lengths of 2.34 and 2.54 Å for the zinc- and mercury-substituted proteins, respectively.

Soft x-ray magnetic circular dichroism on a paramagnetic bioinorganic system

In this paper we report soft X-ray Magnetic Circular Dichroism experiments on paramagnetic bioinorganic systems. We measured the Fe L-edges and the Co L-edges of the Co substituted form of Pyrococcus furiosus rubredoxin, using circularly polarized synchrotron radiation, a split coil superconducting magnet, low sample temperatures, and fluorescence detection. The observed dichroism effects are strong and they can be interpreted by established theoretical procedures. Soft X-ray Magnetic Circular Dichroism demonstrates enormous potential as a new probe for studying paramagnetic bioinorganic systems.