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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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Comparison of the effect of inhibitors on the temperature dependence of phase relaxation rate of the FeS cluster in the FeS motion knockout. Shown are data for ascorbate-reduced (closed symbols) and dithionite-reduced (open symbols) samples of noninhibited cytochrome bc1 (a) and enzyme inhibited with myxothiazol (b), antimycin (c), or stigmatellin (d).
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fig7: Comparison of the effect of inhibitors on the temperature dependence of phase relaxation rate of the FeS cluster in the FeS motion knockout. Shown are data for ascorbate-reduced (closed symbols) and dithionite-reduced (open symbols) samples of noninhibited cytochrome bc1 (a) and enzyme inhibited with myxothiazol (b), antimycin (c), or stigmatellin (d).

Mentions: The correlation between the level of the enhancement and the distance distribution of the FeS head domain is further evident in the experiments performed with the FeS motion knockout. In this form, a+2Ala mutational insertion in the neck region connecting the FeS head domain with the hydrophobic anchor imposes structural constraints and severely impedes the motion arresting the FeS head domain in the Qo site for seconds (20). Thus, as expected, the enhancement of FeS cluster relaxation in this mutant is generally very strong (Figures 7 and 8b). Without any inhibitor added the level of the enhancement even exceeds that observed in the wild-type enzyme treated with stigmatellin (Figure 7a). Addition of antimycin or myxothiazol does not change the level of the enhancement (Figure 7b or 7c, respectively). On the other hand, addition of stigmatellin lowers the level so that it approaches the level of stigmatellin-treated wild-type cytochrome bc1 (Figure 7d).


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)

Comparison of the effect of inhibitors on the temperature dependence of phase relaxation rate of the FeS cluster in the FeS motion knockout. Shown are data for ascorbate-reduced (closed symbols) and dithionite-reduced (open symbols) samples of noninhibited cytochrome bc1 (a) and enzyme inhibited with myxothiazol (b), antimycin (c), or stigmatellin (d).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig7: Comparison of the effect of inhibitors on the temperature dependence of phase relaxation rate of the FeS cluster in the FeS motion knockout. Shown are data for ascorbate-reduced (closed symbols) and dithionite-reduced (open symbols) samples of noninhibited cytochrome bc1 (a) and enzyme inhibited with myxothiazol (b), antimycin (c), or stigmatellin (d).
Mentions: The correlation between the level of the enhancement and the distance distribution of the FeS head domain is further evident in the experiments performed with the FeS motion knockout. In this form, a+2Ala mutational insertion in the neck region connecting the FeS head domain with the hydrophobic anchor imposes structural constraints and severely impedes the motion arresting the FeS head domain in the Qo site for seconds (20). Thus, as expected, the enhancement of FeS cluster relaxation in this mutant is generally very strong (Figures 7 and 8b). Without any inhibitor added the level of the enhancement even exceeds that observed in the wild-type enzyme treated with stigmatellin (Figure 7a). Addition of antimycin or myxothiazol does not change the level of the enhancement (Figure 7b or 7c, respectively). On the other hand, addition of stigmatellin lowers the level so that it approaches the level of stigmatellin-treated wild-type cytochrome bc1 (Figure 7d).

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