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Magnetic interactions sense changes in distance between heme b(L) and the iron-sulfur cluster in cytochrome bc(1).

Sarewicz M, Dutka M, Froncisz W, Osyczka A - Biochemistry (2009)

Bottom Line: The dipolar relaxation curves measured by EPR at Q-band in a glass state of frozen solution (i.e., under the conditions trapping a dynamic distribution of FeS positions that existed in a liquid phase) of isolated cytochrome bc(1) were compared with the curves calculated for the FeS cluster occupying distinct positions in various crystals of cytochrome bc(1).This comparison revealed the existence of a broad distribution of the FeS positions in noninhibited cytochrome bc(1) and demonstrated that the average equilibrium position is modifiable by inhibitors or mutations.To explain the results, we assume that changes in the equilibrium distribution of the FeS positions are the result of modifications of the orienting potential gradient in which the diffusion of the FeS head domain takes place.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland. marcin.sarewicz@gmail.com

ABSTRACT
During the operation of cytochrome bc(1), a key enzyme of biological energy conversion, the iron-sulfur head domain of one of the subunits of the catalytic core undergoes a large-scale movement from the catalytic quinone oxidation Q(o) site to cytochrome c(1). This changes a distance between the two iron-two sulfur (FeS) cluster and other cofactors of the redox chains. Although the role and the mechanism of this movement have been intensely studied, they both remain poorly understood, partly because the movement itself is not easily traceable experimentally. Here, we take advantage of magnetic interactions between the reduced FeS cluster and oxidized heme b(L) to use dipolar enhancement of phase relaxation of the FeS cluster as a spectroscopic parameter which with a unique clarity and specificity senses changes in the distance between those two cofactors. The dipolar relaxation curves measured by EPR at Q-band in a glass state of frozen solution (i.e., under the conditions trapping a dynamic distribution of FeS positions that existed in a liquid phase) of isolated cytochrome bc(1) were compared with the curves calculated for the FeS cluster occupying distinct positions in various crystals of cytochrome bc(1). This comparison revealed the existence of a broad distribution of the FeS positions in noninhibited cytochrome bc(1) and demonstrated that the average equilibrium position is modifiable by inhibitors or mutations. To explain the results, we assume that changes in the equilibrium distribution of the FeS positions are the result of modifications of the orienting potential gradient in which the diffusion of the FeS head domain takes place. The measured changes in the phase relaxation enhancement provide the first direct experimental description of changes in the strength of dipolar coupling between the FeS cluster and heme b(L).

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Effect of temperature and reduction state of heme bL on spin−lattice relaxation rate of the FeS cluster in stigmatellin-treated cytochrome bc1. (a) shows a typical Q-band inversion−recovery curve, registered at 12 K. (b) shows the temperature dependence of spin−lattice relaxation rate in ascorbate- and ditionite-reduced cytochrome bc1 (squares and circles, respectively). Fits in (b) (solid lines) assume dominant Orbach process. Estimated Orbach energies of the FeS cluster for ascorbate- and dithionite-reduced samples are 128 and 133 K, respectively.
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fig3: Effect of temperature and reduction state of heme bL on spin−lattice relaxation rate of the FeS cluster in stigmatellin-treated cytochrome bc1. (a) shows a typical Q-band inversion−recovery curve, registered at 12 K. (b) shows the temperature dependence of spin−lattice relaxation rate in ascorbate- and ditionite-reduced cytochrome bc1 (squares and circles, respectively). Fits in (b) (solid lines) assume dominant Orbach process. Estimated Orbach energies of the FeS cluster for ascorbate- and dithionite-reduced samples are 128 and 133 K, respectively.

Mentions: The spin−lattice relaxation time (T1) of the FeS cluster was determined from the inversion recovery (IR) measurements at temperature range 12−31 K. Fitting the stretched exponent to the IR curve recorded at a given temperature (see Experimental Procedures), such as that shown in Figure 3a, yielded the τww. The spin−lattice relaxation rates 1/T1 calculated from eq 2 were taken to construct the plots of temperature dependence of the relaxation rates. Figure 3b shows the plots obtained with ascorbate- and dithionite-reduced cytochrome bc1 in the presence of stigmatellin. This inhibitor locks the FeS head domain at the Qo position setting the conditions where the distance between the FeS cluster and heme bL is the shortest; thus the strongest influence of this heme can be expected. Yet, the plots revealed no difference between the samples with reduced and oxidized hemes b, indicating that oxidized heme bL does not influence the spin−lattice relaxation rate of the FeS cluster within the tested temperature range.


Magnetic interactions sense changes in distance between heme b(L) and the iron-sulfur cluster in cytochrome bc(1).

Sarewicz M, Dutka M, Froncisz W, Osyczka A - Biochemistry (2009)

Effect of temperature and reduction state of heme bL on spin−lattice relaxation rate of the FeS cluster in stigmatellin-treated cytochrome bc1. (a) shows a typical Q-band inversion−recovery curve, registered at 12 K. (b) shows the temperature dependence of spin−lattice relaxation rate in ascorbate- and ditionite-reduced cytochrome bc1 (squares and circles, respectively). Fits in (b) (solid lines) assume dominant Orbach process. Estimated Orbach energies of the FeS cluster for ascorbate- and dithionite-reduced samples are 128 and 133 K, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2697599&req=5

fig3: Effect of temperature and reduction state of heme bL on spin−lattice relaxation rate of the FeS cluster in stigmatellin-treated cytochrome bc1. (a) shows a typical Q-band inversion−recovery curve, registered at 12 K. (b) shows the temperature dependence of spin−lattice relaxation rate in ascorbate- and ditionite-reduced cytochrome bc1 (squares and circles, respectively). Fits in (b) (solid lines) assume dominant Orbach process. Estimated Orbach energies of the FeS cluster for ascorbate- and dithionite-reduced samples are 128 and 133 K, respectively.
Mentions: The spin−lattice relaxation time (T1) of the FeS cluster was determined from the inversion recovery (IR) measurements at temperature range 12−31 K. Fitting the stretched exponent to the IR curve recorded at a given temperature (see Experimental Procedures), such as that shown in Figure 3a, yielded the τww. The spin−lattice relaxation rates 1/T1 calculated from eq 2 were taken to construct the plots of temperature dependence of the relaxation rates. Figure 3b shows the plots obtained with ascorbate- and dithionite-reduced cytochrome bc1 in the presence of stigmatellin. This inhibitor locks the FeS head domain at the Qo position setting the conditions where the distance between the FeS cluster and heme bL is the shortest; thus the strongest influence of this heme can be expected. Yet, the plots revealed no difference between the samples with reduced and oxidized hemes b, indicating that oxidized heme bL does not influence the spin−lattice relaxation rate of the FeS cluster within the tested temperature range.

Bottom Line: The dipolar relaxation curves measured by EPR at Q-band in a glass state of frozen solution (i.e., under the conditions trapping a dynamic distribution of FeS positions that existed in a liquid phase) of isolated cytochrome bc(1) were compared with the curves calculated for the FeS cluster occupying distinct positions in various crystals of cytochrome bc(1).This comparison revealed the existence of a broad distribution of the FeS positions in noninhibited cytochrome bc(1) and demonstrated that the average equilibrium position is modifiable by inhibitors or mutations.To explain the results, we assume that changes in the equilibrium distribution of the FeS positions are the result of modifications of the orienting potential gradient in which the diffusion of the FeS head domain takes place.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland. marcin.sarewicz@gmail.com

ABSTRACT
During the operation of cytochrome bc(1), a key enzyme of biological energy conversion, the iron-sulfur head domain of one of the subunits of the catalytic core undergoes a large-scale movement from the catalytic quinone oxidation Q(o) site to cytochrome c(1). This changes a distance between the two iron-two sulfur (FeS) cluster and other cofactors of the redox chains. Although the role and the mechanism of this movement have been intensely studied, they both remain poorly understood, partly because the movement itself is not easily traceable experimentally. Here, we take advantage of magnetic interactions between the reduced FeS cluster and oxidized heme b(L) to use dipolar enhancement of phase relaxation of the FeS cluster as a spectroscopic parameter which with a unique clarity and specificity senses changes in the distance between those two cofactors. The dipolar relaxation curves measured by EPR at Q-band in a glass state of frozen solution (i.e., under the conditions trapping a dynamic distribution of FeS positions that existed in a liquid phase) of isolated cytochrome bc(1) were compared with the curves calculated for the FeS cluster occupying distinct positions in various crystals of cytochrome bc(1). This comparison revealed the existence of a broad distribution of the FeS positions in noninhibited cytochrome bc(1) and demonstrated that the average equilibrium position is modifiable by inhibitors or mutations. To explain the results, we assume that changes in the equilibrium distribution of the FeS positions are the result of modifications of the orienting potential gradient in which the diffusion of the FeS head domain takes place. The measured changes in the phase relaxation enhancement provide the first direct experimental description of changes in the strength of dipolar coupling between the FeS cluster and heme b(L).

Show MeSH
Related in: MedlinePlus