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The structure of a partially deuterated rubredoxin from the hyperthermophilic archaeon Pyrococcus furiosus, an organism that grows optimally at 100[degrees]C, was determined by using the neutron single-crystal diffractometer dedicated for biological macromolecules (BIX-3) at the JRR-3M reactor of the Japan Atomic Energy Research Institute. Data were collected at room temperature up to a resolution of 1.5 [Angstrom], and the completeness factor of the data set was 81.9%. The model contains 306 H and 50 D atoms. A total of 37 hydration water molecules were identified, with 15 having all three atoms fully located and the remaining [D.sub.2]O molecules partially defined. The model has been refined to final agreement factors of R = 18.6% and [R.sub.free] = 21.7%. Several orientations of the O-D bonds of side chains, whose assignments from x-ray data were previously ambiguous, were clearly visible in the neutron structure. Although most backbone N-H bonds had undergone some degree of H/D exchange throughout the rubredoxin molecule, 5 H atom positions still had distinctly negative (H) peaks. The neutron Fourier maps clearly showed the details of an extensive set of H bonds involving the [ND.sub.3.sup.+] terminus that may contribute to the unusual thermostability of this molecule.

Nitric oxide (NO) has been used as a substrate analog to explore the structural and electronic determinants of enzymatic superoxide reduction at the mononuclear iron active site of Pyrococcus furiosus superoxide reductase (SOR) through the use of EPR, resonance Raman, Fourier transform IR, UV-visible absorption, and variable-temperature variable-field magnetic CD spectroscopies. The NO adduct of reduced SOR is shown to have a near-axial S = 3/2 ground state with E/D = 0.06 and D = 12 [+ or -] 2 [cm.sup.-1] (where D and E are the axial and rhombic zero-field splitting parameters, respectively) and the UV-visible absorption and magnetic CD spectra are dominated by an out-of-plane N[O.su.-]([[pi].sup.*])-to-[Fe.sup.3+](d[pi]) charge-transfer transition, polarized along the zero-field splitting axis. Resonance Raman studies indicate that the NO adduct is six-coordinate with NO ligated in a bent conformation trans to the cysteinyl S, as evidenced by the identification of v(N-O) at 1,721 [cm.sup.-1], v(Fe-NO) at 475 [cm.sup.-1], and v(Fe-S(Cys), at 291 [cm.sup.-1], via [sup.34]S and [sup.15]NO isotope shifts. The electronic and vibrational properties of the S = 3/2 [{FeNO}.sup.7] unit are rationalized in terms of a limiting formulation involving a high-spin (S = 5/2) [Fe.sup.3+] center antiferromagnetically coupled to a (S = 1) N[O.sup.-] anion, with a highly covalent [Fe.sup.3+]-N[O.sup.-] interaction. The results support a catalytic mechanism for SOR, with the first step involving oxidative addition of superoxide to form a ferric-peroxo intermediate, and indicate the important roles that the Fe spin state and the trans cysteinate ligand play in effecting superoxide reduction and peroxide release.

Rubredoxin from the hyperthermophile Pyrococcus furiosus is the most thermostable protein characterized to date with an estimated global unfolding rate of [10.sup.-6] [s.sup.-1] at 100 [degrees] C. In marked contrast to these slow global dynamics, hydrogen exchange experiments here demonstrate that conformational opening for solvent access occurs in the [approximately equals] millisecond time frame or faster at 28 [degrees] C for all amide positions. Under these conditions all backbone amides with exchange protection factors between [10.sup.4] and [10.sup.6], for which [EX.sub.2] exchange kinetics were directly verified, have exchange activation energy values within 2-3 kcal/mol of that observed for unstructured peptides. The conformational flexibility of this protein is thus sufficient for water and base catalyst access to the exchanging amide with quite limited structural disruption. The common hypothesis that enhanced conformational rigidity in the folded native state underlies the increased thermal stability of hyperthermophile proteins is not supported by these data.