The 3Fe forms of ferredoxins (Fd) from the hyperthermophilic archaebacteria Pyrococcus furiosus (Pf) and Thermococcus litoralis (Tl) have been investigated by 1H NMR. A combination of one-dimensional nuclear Overhauser and two-dimensional NOESY and bond correlation spectroscopy provides the assignment of the aromatic residues, one conserved valine, and the location of the signals for each of the three cysteines coordinated to the clusters. Dipolar contacts between the Trp 2 and Tyr 46 in Pf Fd and from an invariant phenylalanine to an invariant valine and a cluster cysteine in both Fd confirm a folding pattern for these proteins that is very similar to that of the crystallographically characterized ferredoxin from the mesophile Desulfovibro gigas. The sequence-specific assignment of the buried cysteine near the invariant phenylalanine has been made. The temperature dependence of the contact-shifted cysteinyl residues reveals a distinct 2:1 asymmetry in the magnetic coupling among the three high-spin ferric ions, in that one cysteine exhibits Curie behavior, while the other two cysteines display anti-Curie behavior. These magnetic properties are rationalized qualitatively on the basis of a magnetic coupling scheme where two iron couple to yield an intermediate spin of 2 which couples to the remaining S = 5/2 iron to yield the total cluster spin 1/2. This magnetic asymmetry appears to be a characteristic feature of oxidized 3 Fe clusters. Pf Fd also undergoes a dynamic equilibrium between two alternate forms that differ slightly in the environment of two of the coordinated cysteines. Analysis of the pattern of the contact shifts for the three cysteines in the two ferredoxins suggests that the cysteine coordinated to the unique iron does not have the same sequence origin.

The reaction of heme and apoprotein has been studied in detail in 1H NMR spectroscopy in order to elucidate the conditions for reconstitution of hemoglobin (Hb) to yield the native protein. The initially formed holoprotein exists as a mixture of isomers with individual subunits possessing the two heme orientations differing by a 180 degrees rotation about the alpha, gamma-meso axis [La Mar, G. N., Yamamoto, Y., Jue, T., Smith, K. M., & Pandey, R. K. (1985) Biochemistry 24, 3826-3831]. We characterize in detail herein the rates and mechanism of heme reorientation and show that the rates differ dramatically for met-aquo and met-azido derivatives and are highly pH dependent in both subunits in a fashion that allows selective equilibration in either subunit. Nonequilibrium mixtures of such isomers can be kinetically trapped in the met-azido form and stored in this metastable form for many months. With kinetically controlled heme orientationally disordered Hb, unambiguous assignment of 1H NMR resonances to individual subunits has been made for the met-azido derivative, which demonstrates approximately 2% and 10% equilibrium heme disorder in the alpha- and beta-subunits, respectively. Comparison of the 1H NMR spectra of various heme rotationally disordered Hb derivatives indicates that this disorder is observable in all forms studied, but is most easily recognized as heme disorder and most conveniently monitored in the met-azido complex. Structural consequences of heme disorder appear to manifest themselves much more strongly in peripheral than axial interactions at the heme. Preliminary studies reveal that both the rate of autoxidation of oxy-Hb and the azide affinity of met-aquo-Hb depend on the orientation of the heme.

The oxidized and reduced forms of the [4Fe-4S]-containing ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus, Pf, have been investigated by 1H nuclear magnetic resonance spectroscopy, electron paramagnetic resonance spectroscopy and thiol titrations. We have identified and isolated at Ambient temperature four distinct redox states for the [4Fe-4S] form of the ferredoxin. These states differ in the redox state of the cluster, which is coordinated by Cys 11, Asp 14, Cys 17, and Cys 56, and of a disulfide bridge between Cys 21 and Cys 48. The protein, as isolated under anaerobic conditions, designated 4Fe FdBred, contains the reduced cluster and two free thiols. The cluster, but not the thiols, is readily oxidized by brief exposure to O2 to yield 4Fe FdBOX. Prolonged O2 treatment (> 24 h at 30 degrees C) is required to generate the protein with a disulfide (4Fe FdAOX) while this fully oxidized form is readily converted by brief reduction with sodium dithionite to the protein with a reduced cluster and a disulfide (4Fe FdAred). Analyses of the magnitude and the number of hyperfine-shifted resonances in each of the four redox states are discussed.

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