Monitoring mobility in the early steps of unfolding: the case of oxidized cytochrome b(5) in the presence of 2 M guanidinium chloride.

A Model-Free analysis of the (15)N relaxation properties of oxidized cytochrome b(5), a heme-containing electron-transfer protein, has been performed in 2 M guanidinium chloride (GdmCl), i.e., just before the heme is released by the action of denaturant. This analysis provides information on the mobility in the nano- to picosecond time range. A parallel study on the motions in the milli- to microsecond time scale has also been performed by analyzing rotating-frame (15)N relaxation rates. The protein contains a 60:40 ratio of two conformers (A and B) differing for the rotation of the heme group around the alpha-gamma meso axis. The effect of denaturant has been followed for both species, and the mobility properties have been compared with the analogous information in the absence of denaturant. To complete the picture, we also performed (15)N relaxation measurements and the Model-Free analysis of the native B form, whereas data on the A form [Dangi, B., Sarma, S., Yan, C., Banville, D. L., Guiles, R. D. (1998) J. Phys. Chem. B 102, 8201-8208], as well as rotating-frame measurements for both native forms [Banci, L., Bertini, I., Cavazza, C., Felli, I. C., Koulougliotis, D. (1998) Biochemistry 37, 12320-12330; Arnesano, F., Banci, L., Bertini, I., Felli, I. C., Koulougliotis, D. (1999) Eur. J. Biochem. 260, 347-354], are already available in the literature. It is found that GdmCl tends to increase the internal mobility, although some residues are rigidified on both time scales. In the milli- to microsecond time scale, the tendency to increased mobility is reflected in a decrease in the tau(ex) values rather than in the number of residues experiencing conformational equilibria. In the nano- to picosecond time scale, the tendency to increased mobility is indicated by an overall decrease in the S(2) values. Color pictures are reported to visually show these effects. On the fast time scale, the B form is more mobile than the A form, reflecting the different stability with respect to unfolding. The increase in mobility upon addition of denaturant largely occurs around the heme pocket, which facilitates the release of the heme. The relevance of the internal motions with respect to the early steps of the unfolding process is also analyzed and discussed.

Solution structure of the B form of oxidized rat microsomal cytochrome b5 and backbone dynamics via 15N rotating-frame NMR-relaxation measurements. Biological implications.

Cytochrome b5 in solution has two isomers (A and B) differing by a 180 degrees rotation of the protoporphyrin IX plane around the axis defined by the alpha and gamma meso protons. Homonuclear and heteronuclear NMR spectroscopy has been employed in order to solve the solution structure of the minor (B) form of the oxidized state of the protein and to probe its backbone dynamics in the microsecond--ms timescale in both oxidation states. A family of 40 conformers has been obtained using 1302 meaningful NOEs and 220 pseudocontact shifts and is characterized by high quality and good resolution (rmsd to the mean structure of 0.055 +/- 0.009 nm and 0.103 +/- 0.011 nm for backbone and heavy atoms, respectively). Extensive comparisons of the structural and dynamics changes associated with the A-to-B form interconversion for both oxidation states were subsequently performed. Propionate 6 experiences a redox-state-dependent reorientation as does propionate 7 in the A form. Significant insights are obtained into the role of the protein frame for efficient biological function and backbone mobility is proposed to be one of the factors that could control the reduction potential of the heme.

Solution structure of oxidized rat microsomal cytochrome b5 in the presence of 2 M guanidinium chloride: monitoring the early steps in protein unfolding.

One- and two-dimensional proton NMR spectroscopy has been employed in order to study the denaturation effect of guanidinium chloride (GdmCl) on the oxidized state of the A-form of rat microsomal cytochrome b5 (cyt b5). The protein rapidly starts losing the heme at denaturant concentrations larger than approximately 2.0 M and a largely unfolded protein is eventually obtained. An estimate of the unfolding kinetics is obtained and, by use of a two-state model (folded left and right arrow unfolded), a value for DeltaG degrees. Below this concentration, small (

Identification of slow motions in the reduced recombinant high-potential iron sulfur protein I (HiPIP I) from Ectothiorhodospira halophila via 15N rotating-frame NMR relaxation measurements.

Rotating-frame 15N relaxation rate (R1 rho) NMR experiments have been performed in order to study the dynamic behavior of the reduced recombinant high-potential iron-sulfur protein iso I (HiPIP I) from Ectothiorhodospira halophila, in the microsecond to ms time range. Measurements of R1 rho were performed as a function of the effective spinlock magnetic field amplitude by using both on and off-resonance radio frequency irradiation. The two data sets provided consistent results and were fit globally in order to identify possible exchange processes in an external loop of the reduced HiPIP I. The loop consists of residues 43-45 and the correlation time of the exchange process was determined to be 50 +/- 8 microseconds for the backbone nitrogen of Gln 44.

Probing the backbone dynamics of oxidized and reduced rat microsomal cytochrome b5 via 15N rotating frame NMR relaxation measurements: biological implications.

Rotating frame 15N relaxation NMR experiments have been performed to study the local mobility of the oxidized and reduced forms of rat microsomal cytochrome b5, in the microsecond to millisecond time range. Measurements of rotating frame relaxation rates (R1rho) were performed as a function of the effective magnetic field amplitude by using off-resonance radio frequency irradiation. Detailed analysis of the two data sets resulted in the identification of slow motions along the backbone nitrogens for both oxidation states of the protein. The local mobility of reduced and oxidized cytochrome b5 turned out to be significantly different; 28 backbone nitrogens of the oxidized form were shown to participate in a conformational exchange process, while this number dropped to 12 in the reduced form. The correlation time, tauex, for the exchange processes could be determined for 21 and 9 backbone nitrogens for oxidized and reduced cytochrome b5, respectively, with their values ranging between 70 and 280 microseconds. The direct experimental evidence provided in this study for the larger mobility of the oxidized form of the protein is consistent with the different backbone NH solvent exchangeability recently documented for the two oxidation states [Arnesano, F., et al. (1998) Biochemistry 37, 173-184]. Our experimental observations may have significant biological implications. The differential local mobility between the two oxidation states is proposed to be an important factor controlling the molecular recognition processes in which cytochrome b5 is involved.

Solution structure of oxidized cytochrome c6 from the green alga Monoraphidium braunii.

Cytochrome c6 from Monoraphidium braunii, an 89-amino acid electron transfer protein, has been investigated by NMR in solution, in its oxidized form, at pH 7 and 300 K. By using a combination of COSY, TOCSY, and NOESY experiments, 84% of the proton resonances have been assigned. A total of 1668 experimental NOE constraints, 1109 of which were meaningful, together with 288 pseudocontact shifts, have been used to determine the structure in solution. This is represented as a family of 40 structures which have been energy minimized. The rmsd values with respect to the mean structure are 0.57 +/- 0.08 and 0.94 +/- 0.09 A for the backbone and heavy atoms, respectively. The structure has been found to be very similar to that of the reduced form, except for a rearrangement in propionate 7, a feature which has been observed in all c-type cytochromes investigated so far. Such a feature could be relevant for the efficiency of the electron transfer pathway with either the oxidizing or the reducing partners. Other differences in the oxidation states have been noted in the region proposed to be involved in the interaction with the physiological partners.

The tetranuclear manganese cluster in photosystem II: location and magnetic properties of the S2 state as determined by saturation-recovery EPR spectroscopy.

The spin-lattice relaxation enhancement of the dark-stable tyrosine radical, YD., by the S2 state of the O2-evolving complex (OEC) of photosystem II (PSII) has been measured by using saturation-recovery EPR spectroscopy. Two forms of the S2 state have been compared: the multiline EPR signal species in untreated PSII and the altered multiline EPR signal species in NH3-treated PSII. Previous work has shown that the non-single-exponential spin-lattice relaxation kinetics of YD. in S2-state PSII result from a dipole-dipole interaction with the Mn4 cluster of the OEC. By taking into account the temperature variation of the effective magnetic moment of the S2-state multiline EPR signal form of the OEC, we provide a quantitative analysis of its temperature-dependent enhancement of the spin-lattice relaxation of YD.. Different spin states of the Mn4 cluster in the S2 state are responsible for the effect at different temperature regimes: for T /= 30 K, it is the first excited spin state; and at intermediate temperatures, the contributions of the two spin states are comparable. The relaxation enhancement of YD. is equivalent for both forms of the S2-state multiline EPR signal examined, indicating that the magnetic properties of the Mn4 cluster are very similar in the S2 state for both untreated and NH3-treated PSII. EPR progressive microwave-power saturation has also been used to assess the spin-lattice relaxation properties of the Mn4 cluster giving the altered S2-state multiline EPR signal in the NH3 derivative of PSII. The Orbach mechanism is shown to provide the dominant relaxation pathway; the energy difference between the ground and first excited spin states is estimated to be 30 +/- 2 cm-1, which is very similar to the value found for the S2-state multiline EPR signal species in untreated PSII. Below 4 K, the effectiveness of the S2-state multiline EPR signal species as a spin relaxation enhancer of YD. drops dramatically. This is interpreted to occur because of temperature-dependent 55Mn nuclear spin-lattice relaxation which causes averaging of the effective Larmor frequency of the S2-state multiline EPR signal species during the time scale for spin-lattice relaxation of YD.; because the line shape of the S2-state multiline EPR signal is dominated by isotropic 55Mn nuclear hyperfine splittings, such nuclear relaxation processes allow frequencies in near resonance with that of YD. to be accessed, thereby producing a greater relaxation enhancement. By using a dipolar model that includes the line shapes of both the YD. and S2-state multiline EPR signals, the spin-lattice relaxation enhancement of YD. is analyzed to obtain a lower limit of 22 A for the distance between YD. and the OEC. Together with recent studies showing a close proximity of the Mn4 cluster to YZ., these results provide further support for an asymmetric location of the Mn4 cluster with respect to the two redox-active tyrosines in PSII.

Spectroscopic evidence for the symmetric location of tyrosines D and Z in photosystem II.

Saturation-recovery EPR spectroscopy has been used to probe the location of the redox-active tyrosines, YD (tyrosine 160 of the D2 polypeptide, cyanobacterial numbering) and YZ (tyrosine 161 of the D1 polypeptide), relative to the non-heme Fe(II) in Mn-depleted photosystem II (PSII). Measurements have been made on PSII membranes isolated from spinach and on PSII core complexes purified from the cyanobacterium Synechocystis sp. PCC 6803. In the case of Synechocystis PSII, site-directed mutagenesis of the YD residue to either phenylalanine (Y160F) or methionine (Y160M) was done to eliminate the dark-stable YD.species and, thereby, allow direct spectroscopic observation of the YZ. EPR signal. The spin-lattice relaxation transients of both YD. and YZ. were non-single-exponential due to a dipolar interaction with one of the other paramagnetic species in PSII. Measurements on CN(-)-treated, Mn-depleted cyanobacterial PSII, in which the non-heme Fe(II) was converted into its low-spin, diamagnetic state, proved that the non-heme Fe(II) was the sole spin-lattice relaxation enhancer for both the YD. and YZ. radicals. This justified the use of a dipolar model in order to fit the saturation-recovery EPR data, which were taken over the temperature range 4-70 K. The dipolar rate constants extracted from the fits were identical in magnitude and had the same temperature dependence for both YD. and YZ.. The observation of identical dipolar interactions between YD. and YZ. and the non-heme Fe(II) shows that the distance from each tyrosine to the non-heme Fe(II) is the same.(ABSTRACT TRUNCATED AT 250 WORDS)

Location of chlorophyllZ in photosystem II.

Saturation-recovery and progressive microwave power saturation EPR spectroscopies have been used to probe the location of the chlorophyllZ+ (ChlZ+) radical species in Mn-depleted photosystems II (PSII). The spin-lattice relaxation transients of ChlZ+ were non-single-exponential due to a dipole-dipole interaction with one of the other paramagnetic centers in PSII. Measurements on CN(-)-treated, Mn-depleted PSII membrane samples, in which the non-heme Fe(II) is converted into its low-spin, diamagnetic form, confirmed that the non-heme Fe(II) caused the dipolar relaxation enhancement of ChlZ+. The saturation-recovery EPR data were fit to a dipolar model [Hirsh, D. J., Beck, W. F., Innes, J. B., & Brudvig, G. W. (1992) Biochemistry 31, 532] which takes into account the isotropic (scalar) and orientation-dependent (dipolar) contributions to the spin-lattice relaxation of the radical. The temperature dependence of the dipolar rate constants of ChlZ+ was identical to the temperature dependencies recently observed for the stable tyrosine radical, YD., and the special pair bacteriochlorophyll radical, (BChla)2+, in PSII and in reaction centers from Rhodobacter sphaeroides, respectively. Because the non-heme Fe(II) is known to cause a dipolar relaxation enhancement of the radicals in both of the latter cases, this result provides further evidence that the non-heme Fe(II) causes the dipolar relaxation enhancement of ChlZ+ and, moreover, demonstrates that the magnetic properties of the non-heme Fe(II) in PSII and in reaction centers from Rhodobacter sphaeroides are very similar. By using the known Fe(II)-(BChla)2+ distance for calibration, we estimate the Fe(II)-ChlZ+ distance to be 39.5 +/- 2.5 A.(ABSTRACT TRUNCATED AT 250 WORDS)