Backbone dynamics of plastocyanin in both oxidation states. Solution structure of the reduced form and comparison with the oxidized state.

A model-free analysis based on (15)N R(1), (15)N R(2), and (15)N-(1)H nuclear Overhauser effects was performed on reduced (diamagnetic) and oxidized (paramagnetic) forms of plastocyanin from Synechocystis sp. PCC6803. The protein backbone is rigid, displaying a small degree of mobility in the sub-nanosecond time scale. The loops surrounding the copper ion, involved in physiological electron transfer, feature a higher extent of flexibility in the longer time scale in both redox states, as measured from D(2)O exchange of amide protons and from NH-H(2)O saturation transfer experiments. In contrast to the situation for other electron transfer proteins, no significant difference in the dynamic properties is found between the two redox forms. A solution structure was also determined for the reduced plastocyanin and compared with the solution structure of the oxidized form in order to assess possible structural changes related to the copper ion redox state. Within the attained resolution, the structure of the reduced plastocyanin is indistinguishable from that of the oxidized form, even though small chemical shift differences are observed. The present characterization provides information on both the structural and dynamic behavior of blue copper proteins in solution that is useful to understand further the role(s) of protein dynamics in electron transfer processes.

The first solution structure of a paramagnetic copper(II) protein: the case of oxidized plastocyanin from the cyanobacterium Synechocystis PCC6803.

The NMR solution structure of oxidized plastocyanin from the cyanobacterium Synechocystis PCC6803 is here reported. The protein contains paramagnetic copper(II), whose electronic relaxation times are quite unfavorable for NMR solution studies. The structure has been solved on the basis of 1041 meaningful NOESY cross-peaks, 18 1D NOEs, 26 T(1) values, 96 dihedral angle constraints, and 18 H-bonds. The detection of broad hyperfine-shifted signals and their full assignment allowed the identification of the copper(II) ligands and the determination of the Cu-S-C-H dihedral angle for the coordinated cysteine. The global root-mean-square deviation from the mean structure for the solution structure family is 0.72 +/- 0.14 and 1.16 +/- 0.17 A for backbone and heavy atoms, respectively. The structure is overall quite satisfactory and represents a breakthrough, in that it includes paramagnetic copper proteins among the metalloproteins for which solution structures can be afforded. The comparison with the available X-ray structure of a triple mutant is also performed.

Isolation and physico-chemical characterization of a cytochrome c from the methylotrophic yeast Hansenula polymorpha.

Cytochrome c from the methylotrophic yeast Hansenula polymorpha was isolated and purified to homogeneity for the first time. The final yield of the highly purified protein from 1.4 kg (wet weight) cells was about 20 mg. The hemoprotein has an apparent molecular mass of 12 kDa and isoelectric point (pI) of 9.3. The purified protein was characterized by electronic, EPR and NMR spectroscopies. The redox potential of the cytochrome, E degrees, measured by cyclic voltammetry measurements at neutral pH, is 0.302 V. Both NMR spectroscopy and electrochemical measurements confirm the presence in the solution of several acid-base equilibria, the most pronounced being characterized by a pK(a) of 8.3. The latter pK(a) was attributed to the detachment of the iron(III) ion-coordinated methionine and its replacement by a lysine residue. The electrochemically derived thermodynamic parameters for neutral and alkaline protein species (DeltaS degrees (rc) and DeltaH degrees (rc)) were obtained from the temperature dependence of the redox potential.

An NMR study of the 7Fe-8S ferredoxin from Rhodopseudomonas palustris and reinterpretation of data on similar systems.

The oxidized 7Fe-8S ferredoxin from Rhodopseudomonas palustris is shown to possess a unique 1H NMR spectrum displaying at least one hyperfine-shifted beta-CH2 signal for each cysteine bound to the [3Fe-4S] cluster. COSY and TOCSY spectra and 1- and 2-dimensional NOE experiments, in conjunction with a thorough reexamination of the 1H NMR data on similar systems, permitted the sequential assignment of all of the cysteine beta-CH2 protons even in the absence of the amino acid sequence. The sequential assignment stems on the homology of the hyperfine shift pattern with those of other sequenced 7Fe-8S ferredoxins, which points to a substantial homology in tertiary structure. From the assignment, an analysis of the antiferromagnetic coupling in the [3Fe-4S] system was performed on the basis of a general model of exchange coupling. The NMR signal patterns of [3Fe-4S] clusters in both 3Fe-4S and 7Fe-8S ferredoxins have been discussed, and some correlations are proposed between signal patterns and the primary sequence.

Charge reversal of a critical active-site residue of cytochrome-c peroxidase: characterization of the Arg48-->Glu variant.

A new variant of cytochrome-c peroxidase in which the positively charged Arg48 present in the distal heme-binding pocket has been replaced with a Glu residue has been prepared and characterized to explore, in part, the possibility that a negative charge close to the heme could contribute to stabilization of a porphyrin-centered pi-cation radical in the compound I derivative of the variant. Between pH 4 and 8, this variant forms three pH-linked spectroscopic species. The electronic absorption and 1H-NMR spectra of the predominant form at low pH (HS1) are indicative of a high-spin, pentacoordinate heme iron system. Near neutral pH, a second high-spin species (HS2) is dominant, in which the heme iron center is hexacoordinated, with a water molecule as the sixth axial ligand. At high pH, the third form (LS) exhibits the spectroscopic characteristics of a low-spin, hexacoordinate heme center with bishistidine axial ligation. The apparent pKa values for these transitions are 4.4 and 7.4, respectively, in phosphate buffers and 5.0 and 7.1, respectively, in phosphate/nitrate buffers. Replacement of Arg48 with Glu reduces the thermal stability of the enzyme and also decreases the Fe(III)/Fe(II) reduction potential of the enzyme by approximately 50 mV relative to that of the wild-type enzyme. The stability of compound I formed by the variant is decreased although the rate at which it forms is just one order of magnitude less than that of the wild-type enzyme, thus confirming previous results which indicate that the function of residue 48 in the wild-type peroxidase is more related to the stability of compound I than to its formation [Erman, J. E., Vitello, L. B., Miller, M. A. & Kraut, J. (1992) J. Am. Chem. Soc. 114, 6592-6593; Vitello, L. B., Erman, J. E., Miller, M. A., Wang, J. & Kraut, J. (1993) Biochemistry 32, 9807-9818]. Stopped-flow studies failed to detect even transient formation of a porphyrin-centered radical following addition of hydrogen peroxide to the Fe(III)-enzyme. The consequences of this drastic electrostatic modification of the active site on the steady-state kinetics of the variant are relatively minor.

Three-dimensional structure of the reduced C77S mutant of the Chromatium vinosum high-potential iron-sulfur protein through nuclear magnetic resonance: comparison with the solution structure of the wild-type protein.

The full 1H NMR assignment of the reduced C77S mutant of Chromatium vinosum high-potential iron-sulfur protein (HiPIP) was achieved by taking advantage of the assignment available for the wild-type protein. A total of 1565 nuclear Overhauser effect (NOE) spectroscopy cross peaks were integrated and converted into distance constraints, of which 497 were found to be irrelevant. An additional 24 dipolar constraints were obtained from one-dimensional NOE difference spectra by saturating hyperfine-shifted beta CH2 cysteine/serine protons. Forty-six 3JNH-H alpha coupling constants and eight hydrogen bonds provided further constraints. Through a distance geometry approach, a family of 15 structures was calculated, which was subsequently subjected to restrained energy minimization. The root mean square deviations of the minimized structures were 0.62 +/- 0.09 and 1.09 +/- 0.11 A for backbone and heavy atoms, respectively. The resulting solution structures are very similar to those of the reduced wild-type protein (WT). An analysis of the NOEs experienced by the protons of Ser-77 in both the reduced and oxidized forms reveals that they are very similar to those experienced by Cys-77 in WT. On the basis of the hyperfine shifts observed for the Ser-77 protons and of the present structural analysis, it is concluded that the serine O gamma atom is coordinated to the polymetallic center, thus confirming the strict analogy of the electronic structures of the polymetallic center in both proteins. Capillary electrophoresis experiments demonstrate coordination of Ser-77 as an anion. Serine versus cysteine coordination in iron-sulfur proteins is briefly discussed.

Evidence of histidine coordination to the catalytic ferrous ion in the ring-cleaving 2,2',3-trihydroxybiphenyl dioxygenase from the dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1.

The 1H NMR spectra of an aromatic ring-cleaving extradiol dioxygenase, 2,2',3-trihydroxybiphenyl dioxygenase of the dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1, are reported. In the catalytically active reduced form of the monomeric enzyme (MW = 32 kDa), three broad strongly downfield shifted signals were observed, two of which disappeared in D2O solution. Their shifts and linewidths are consistent with ring NH and meta-like protons of coordinated histidines. These signals show strong sensitivity to the presence of the substrate. The oxidized form of the enzyme shows no hyperfine shifted signals. It is suggested that the high spin Fe(II) ion present in the active form of the enzyme is coordinated by at least two histidines. This is the first report of hyperfine shifted NMR signals being detected for an extradiol dioxygenase.

Three-dimensional solution structure of the oxidized high potential iron-sulfur protein from Chromatium vinosum through NMR. Comparative analysis with the solution structure of the reduced species.

The NMR solution structure of the oxidized HiPIP from Chromatium vinosum has been solved. Despite the fact that the protein is paramagnetic, 85% of the 1H and 80% of the 15N signals have been assigned. Through 1537 NOEs, out of which 1142 were found to be relevant for the structure determination, a family of structures has been obtained by distance geometry calculations. These structures have then been subjected to restrained energy minimization (REM) and restrained molecular dynamics (RMD) calculations in vacuum. Finally, the mean structure of the RMD family has been treated through RMD in water. The RMSD values for the backbone and heavy atoms within the RMD family are 0.57 +/- 0.14 and 1.08 +/- 0.16 A, respectively. These values together with other parameters indicate that the structure is of good quality and as good as the structure of the reduced protein. The RMDw structures of the reduced and oxidized proteins are different beyond the experimental indetermination. The set of constraints for the reduced and oxidized forms have been used to treat the available X-ray structure by RMD in water. The two structures generated in this way are quite similar to their respective solution structures, thus confirming that the experimental constraints are capable of yielding two different structures from the same starting structural model. This is the first time that independently determined solution structures of two redox states of a paramagnetic protein are available. Differences between them and the X-ray structure are discussed.

The three-dimensional solution structure of the reduced high-potential iron-sulfur protein from Chromatium vinosum through NMR.

The 1H NMR assignment of the reduced HiPIP from Chromatium vinosum available in the literature [Gaillard, J., Albrand, J.-P., Moulis, J.-M., & Wemmer, D. E. (1992) Biochemistry 31, 5632-5639] has been extended up to 85% of the total protein protons. Ninety percent of the nitrogens have been assigned. Then the solution structure has been obtained using as many as 1147 meaningful NOE connectivities. The protein is sizably paramagnetic even though the ground state is a singlet. Nevertheless, the final RMSD values are 0.62 and 1.19 A for the backbone and the heavy atoms, respectively. These values compare well with those for diamagnetic proteins of the same size. The solution structure is discussed in the light of the available structural information from X-ray data.

1H one-dimensional and two-dimensional NMR studies of the ferricytochrome c 551 from Rhodocyclus gelatinosus.

1H two-dimensional NMR spectroscopy has been applied to the oxidized form of cytochrome c 551 from Rhodocyclus gelatinosus, which is paramagnetic with S = 1/2. The investigation has allowed a complete and unambiguous assignment of the heme protons and some residues around the heme. We have learned that: the conformation of the axial methionine is equal to that of horse heart cytochrome c and different from two isoenzymes of the same cytochrome c 551 from a different strain; pKa of 6.6 +/- 0.3 has been detected through the shift variations of seventh propionate protons. The detailed differences with other cytochromes c in the hyperfine shifts are discussed.