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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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Temperature dependence of the offset in the dipolar decay curves of native cytochrome bc1 determined for noninhibited (squares) and stigmatellin- (circles) or myxothiazol plus antimycin-treated (triangles) samples.
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fig10: Temperature dependence of the offset in the dipolar decay curves of native cytochrome bc1 determined for noninhibited (squares) and stigmatellin- (circles) or myxothiazol plus antimycin-treated (triangles) samples.

Mentions: A physical meaning can be attributed to this offset when the temperature dependence of dipolar decay is analyzed in the context of the distribution of angles and distances Δ(r, θ) inherent to powder spectra (Figure S2, Supporting Information). A typical decay curve contains the fast- and slow-relaxing components. For the spins with gradually increasing distance r, the dipolar relaxation becomes slower, and their contribution to the tail of the echo decay curve is more significant. Thus, at the point of the experimental cutoff (which, as mentioned above, is different for each temperature) the level of the offset is proportional to the number of slowly relaxing spins (more distant from heme bL). Indeed, the smallest changes in the offset are observed for native enzyme treated with stigmatellin (Figure 10, circles) and the FeS motion knockout (data not shown), which is expected for the sample with a largest population of spins close to heme bL and impacted by the strongest enhancement of relaxation. For other samples the increase in the offset with temperature is larger (Figure 10, squares and triangles), which can be explained by assuming that a population of spins more distant from heme bL becomes larger, leading to an increase in the fraction of slower dipolar decays. This reasoning is supported by the simulations of the dipolar traces. Figure 11 compares the dipolar relaxation curves of the cluster calculated for r = 2.64 nm (Qo position) and r = 3.55 nm (c1 position). Clearly, the position more distant from heme bL (in this case the c1 position) produces greater offset in the dipolar curve if the cutoff of 3000 ns is applied.


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)

Temperature dependence of the offset in the dipolar decay curves of native cytochrome bc1 determined for noninhibited (squares) and stigmatellin- (circles) or myxothiazol plus antimycin-treated (triangles) samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig10: Temperature dependence of the offset in the dipolar decay curves of native cytochrome bc1 determined for noninhibited (squares) and stigmatellin- (circles) or myxothiazol plus antimycin-treated (triangles) samples.
Mentions: A physical meaning can be attributed to this offset when the temperature dependence of dipolar decay is analyzed in the context of the distribution of angles and distances Δ(r, θ) inherent to powder spectra (Figure S2, Supporting Information). A typical decay curve contains the fast- and slow-relaxing components. For the spins with gradually increasing distance r, the dipolar relaxation becomes slower, and their contribution to the tail of the echo decay curve is more significant. Thus, at the point of the experimental cutoff (which, as mentioned above, is different for each temperature) the level of the offset is proportional to the number of slowly relaxing spins (more distant from heme bL). Indeed, the smallest changes in the offset are observed for native enzyme treated with stigmatellin (Figure 10, circles) and the FeS motion knockout (data not shown), which is expected for the sample with a largest population of spins close to heme bL and impacted by the strongest enhancement of relaxation. For other samples the increase in the offset with temperature is larger (Figure 10, squares and triangles), which can be explained by assuming that a population of spins more distant from heme bL becomes larger, leading to an increase in the fraction of slower dipolar decays. This reasoning is supported by the simulations of the dipolar traces. Figure 11 compares the dipolar relaxation curves of the cluster calculated for r = 2.64 nm (Qo position) and r = 3.55 nm (c1 position). Clearly, the position more distant from heme bL (in this case the c1 position) produces greater offset in the dipolar curve if the cutoff of 3000 ns is applied.

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