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.

No abstract

No abstract

The exchange rates of heme cavity histidine nitrogen-bound protons in horse and dog metcyanomyoglobins have been determined at 40 degrees C as a function of pH by 1H NMR spectroscopy. They were compared to the results reported for the sperm whale homologue [Cutnell, J. D., La Mar, G. N., & Kong, S. B. (1981) J. Am. Chem. Soc. 103, 3567-3572]. The rate profiles suggest that the exchange follows EX2-type kinetics, and the relative rate values favor a penetration model over a local unfolding model. It was found that the behavior of protons located on the proximal side of the heme is similar in the three proteins. The distal histidyl imidazole NH, however, shows a highly accelerated hydroxyl ion catalyzed rate in horse and dog myoglobins relative to that in sperm whale myoglobin. NMR spectral and relaxational characteristics of the assigned heme cavity protons indicate that the global geometry of the heme pocket is highly conserved in the ground-state structure of the three proteins. We propose a model that attributes the different distal histidine exchange behavior to the relative dynamic stability of the distal heme pocket in dog or horse myoglobin vs. sperm whale myoglobin. This model involves a dynamic equilibrium between a closed heme pocket as found in metaquomyoglobin [Takano, T. (1977) J. Mol. Biol. 110, 537-568] and an open pocket as found in phenylmetmyoglobin [Ringe, D., Petsko, G. A., Kerr, D. E., & Ortiz de Montellano, P. R. (1984) Biochemistry 23, 2-4].(ABSTRACT TRUNCATED AT 250 WORDS)

Solution two-dimensional 1H NMR studies have been carried out on cyanide-inhibited horseradish peroxidase isozyme C (HRPC-CN) to explore the scope and limitations of identifying residues in the heme pocket and substrate binding site, including those of the "second sphere" of the heme, i.e. residues which do not necessarily have dipolar contact with the heme. The experimental methods use a range of experimental conditions to obtain data on residue protons with a wide range of paramagnetic relaxivity. The signal assignment strategy is guided by the recently reported crystal structure of recombinant HRPC and the use of calculated magnetic axes. The goal of the assignment strategy is to identify signals from all residues in the heme, as well as proximal and distal, environment and the benzhydroxamic acid (BHA) substrate binding pocket. The detection and sequence specific assignment of aromatic and aliphatic residues in the vicinity of the heme pocket confirm the validity of the NMR methodologies described herein. Nearly all residues in the heme periphery are now assigned, and the first assignments of several "second sphere" residues in the heme periphery are reported. The results show that nearly all catalytically relevant amino acids in the active site can be identified by the NMR strategy. The residue assignment strategy is then extended to the BHA:HRPC-CN complex. Two Phe rings (Phe 68 and Phe 179) and an Ala (Ala 140) are shown to be in primary dipolar contact to BHA. The shift changes induced by substrate binding are shown to reflect primarily changes in the FeCN tilt from the heme normal. The present results demonstrate the practicality of detailed solution 1H NMR investigation of the manner in which substrate binding is perturbed by either variable substrates or point mutations of HRP.

The 1H NMR spectrum of the low-spin, cyanide-ligated ferric complex of the myoglobin from the mollusc Aplysia limacina has been investigated. All of the resolved resonances from both the hemin and the proximal histidine have been assigned by a combination of isotope labeling, spin decoupling, analysis of differential paramagnetic relaxation, and nuclear Overhauser (NOE) experiments. The pattern of the heme contact shifts is unprecedented for low-spin ferric hemoproteins in exhibiting minimal rhombic asymmetry. This low in-plane asymmetry is correlated with the X-ray-determined orientation of the proximal histidyl imidazole plane relative to the heme and provides an important test case for the interpretation of hyperfine shifts of low-spin ferric hemoproteins. The bonding of the proximal histidine is shown to be similar to that in sperm whale myoglobin and is largely unperturbed by conformational transitions down to pH approximately 4. The two observed conformational transitions appear to be linked to the titration of the two heme propionate groups, which are suggested to exist in various orientations as a function of both pH and temperature. Heme orientational disorder in the ratio 5:1 was demonstrated by both isotope labeling and NOE experiments. The exchange rate with bulk water of the proximal histidyl labile ring proton is faster in Aplysia than in sperm whale myoglobin, consistent with a greater tendency for local unfolding of the heme pocket in the former protein. A similar increased heme pocket lability in Aplysia myoglobin has been noted in the rate of heme reorientation [Bellelli, A., Foon, R., Ascoli, F.,& Brunori, M. (1987) Biochem. J. 246, 787-789].