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Title: The Raised Midpoint Potential of the[2Fe2S] Cluster of Cytochrome bc1 is Mediated by Both the Q(o) Site Occupants and the Head Domain Position of the Fe-S Protein Subunit

Abstract

We have previously reported that mutant strains of Rhodobacter capsulatus that have alanine insertions (+nAla mutants) in the hinge region of the iron sulfur (Fe-S) containing subunit of the bc1 complex have increased redox midpoint potentials (Em) for their[2Fe2S] clusters. The alteration of the Em in these strains, that contain mutations far from the metal binding site, implied that the local environment of the metal center is indirectly altered by a change in the interaction of this subunit with the hydroquinone oxidizing (Qo) site (Darrouzet, E., Valkova-Valchanova, M., and Daldal, F. (2002) Journal of Biological Chemistry 277, 3464- 3470). Subsequently, the Em changes have been proposed to be predominantly due to a stronger or more stabilized hydrogen bonding between the reduced[2Fe2S] cluster and the Qo site inhabitant ubiquinone (Q) (Shinkarev, V. P., Kolling, D. R. J., Miller, T. J., and Crofts, A. R. (2002) Biochemistry 41, 14372-14382). To further investigate this issue, Fe-S protein-Q interactions were monitored by EPR spectroscopy and the findings indicated that the wild type and mutant proteins interactions with Q are similar. Moreover, when the Qpool was chemically depleted, the Em of the[2Fe2S] cluster in mutant bc1 complexes remained more positive than a similarly treated nativemore » enzyme (e.g., the[2Fe2S] Em of the+2Ala mutant was 55 mV more positive than the wild type). These data suggest that the increased Em of the[2Fe2S] cluster in the+nAla mutants is in part due to the clusters interaction with Q, and in part to additional factors that are independent of hydrogen bonding to Q. One such factor, the possibility of a different position of the Fe-S at the Qo site of the mutant proteins versus the native enzyme, was addressed by determining the orientation of the[2Fe2S] cluster in the membrane using EPR spectroscopy. In the case of the+2Ala mutant, the[2Fe2S] cluster orientation in the absence of inhibitor is different than that seen in the native enzyme. However, the+2Ala mutant cluster shared a similar orientation with the native enzyme when both samples were exposed to either stigmatellin or myxothiazol. In addition, Qpool extracted membranes of+2Ala mutant exhibited fewer overall orientations, with the predominant one being more similar to that observed in the non Q-depleted membranes of+2Ala mutant than the Q-depleted membranes of a wild type strain. Therefore, additional component(s) that are independent of Qo site inhabitants and that originate from the newly observed orientations of the[2Fe2S] clusters in the+nAla mutants also contribute to the increased midpoint potentials of their[2Fe2S] clusters. While the molecular basis of these components remains to be determined, salient implications of these findings in terms of Qo site catalysis are discussed.« less

Authors:
; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Lab., Richland, WA (US), Environmental Molecular Sciences Laboratory (US)
Sponsoring Org.:
US Department of Energy (US)
OSTI Identifier:
15007033
Report Number(s):
PNWD-SA-6308
Journal ID: ISSN 0006-2960; BICHAW; 2358; TRN: US200413%%210
DOE Contract Number:  
AC06-76RL01830
Resource Type:
Journal Article
Journal Name:
Biochemistry (Eaton)
Additional Journal Information:
Journal Volume: 43; Journal Issue: 8; Other Information: PBD: 2 Mar 2004; Journal ID: ISSN 0006-2960
Country of Publication:
United States
Language:
English
Subject:
08 HYDROGEN; ALANINES; BIOCHEMISTRY; BONDING; CATALYSIS; CHEMISTRY; CYTOCHROMES; ENZYMES; HYDROGEN; IRON; MEMBRANES; MUTANTS; MUTATIONS; PROTEINS; SPECTROSCOPY; SULFUR; UBIQUINONE; ENVIRONMENTAL MOLECULAR SCIENCES LABORATORY, NULL

Citation Formats

Cooley, Jason W., Roberts, Arthur G., Bowman, Michael K., Kramer, David M., and Daldal, Fevzi. The Raised Midpoint Potential of the[2Fe2S] Cluster of Cytochrome bc1 is Mediated by Both the Q(o) Site Occupants and the Head Domain Position of the Fe-S Protein Subunit. United States: N. p., 2004. Web. doi:10.1021/bi035938u.
Cooley, Jason W., Roberts, Arthur G., Bowman, Michael K., Kramer, David M., & Daldal, Fevzi. The Raised Midpoint Potential of the[2Fe2S] Cluster of Cytochrome bc1 is Mediated by Both the Q(o) Site Occupants and the Head Domain Position of the Fe-S Protein Subunit. United States. doi:10.1021/bi035938u.
Cooley, Jason W., Roberts, Arthur G., Bowman, Michael K., Kramer, David M., and Daldal, Fevzi. Tue . "The Raised Midpoint Potential of the[2Fe2S] Cluster of Cytochrome bc1 is Mediated by Both the Q(o) Site Occupants and the Head Domain Position of the Fe-S Protein Subunit". United States. doi:10.1021/bi035938u.
@article{osti_15007033,
title = {The Raised Midpoint Potential of the[2Fe2S] Cluster of Cytochrome bc1 is Mediated by Both the Q(o) Site Occupants and the Head Domain Position of the Fe-S Protein Subunit},
author = {Cooley, Jason W. and Roberts, Arthur G. and Bowman, Michael K. and Kramer, David M. and Daldal, Fevzi},
abstractNote = {We have previously reported that mutant strains of Rhodobacter capsulatus that have alanine insertions (+nAla mutants) in the hinge region of the iron sulfur (Fe-S) containing subunit of the bc1 complex have increased redox midpoint potentials (Em) for their[2Fe2S] clusters. The alteration of the Em in these strains, that contain mutations far from the metal binding site, implied that the local environment of the metal center is indirectly altered by a change in the interaction of this subunit with the hydroquinone oxidizing (Qo) site (Darrouzet, E., Valkova-Valchanova, M., and Daldal, F. (2002) Journal of Biological Chemistry 277, 3464- 3470). Subsequently, the Em changes have been proposed to be predominantly due to a stronger or more stabilized hydrogen bonding between the reduced[2Fe2S] cluster and the Qo site inhabitant ubiquinone (Q) (Shinkarev, V. P., Kolling, D. R. J., Miller, T. J., and Crofts, A. R. (2002) Biochemistry 41, 14372-14382). To further investigate this issue, Fe-S protein-Q interactions were monitored by EPR spectroscopy and the findings indicated that the wild type and mutant proteins interactions with Q are similar. Moreover, when the Qpool was chemically depleted, the Em of the[2Fe2S] cluster in mutant bc1 complexes remained more positive than a similarly treated native enzyme (e.g., the[2Fe2S] Em of the+2Ala mutant was 55 mV more positive than the wild type). These data suggest that the increased Em of the[2Fe2S] cluster in the+nAla mutants is in part due to the clusters interaction with Q, and in part to additional factors that are independent of hydrogen bonding to Q. One such factor, the possibility of a different position of the Fe-S at the Qo site of the mutant proteins versus the native enzyme, was addressed by determining the orientation of the[2Fe2S] cluster in the membrane using EPR spectroscopy. In the case of the+2Ala mutant, the[2Fe2S] cluster orientation in the absence of inhibitor is different than that seen in the native enzyme. However, the+2Ala mutant cluster shared a similar orientation with the native enzyme when both samples were exposed to either stigmatellin or myxothiazol. In addition, Qpool extracted membranes of+2Ala mutant exhibited fewer overall orientations, with the predominant one being more similar to that observed in the non Q-depleted membranes of+2Ala mutant than the Q-depleted membranes of a wild type strain. Therefore, additional component(s) that are independent of Qo site inhabitants and that originate from the newly observed orientations of the[2Fe2S] clusters in the+nAla mutants also contribute to the increased midpoint potentials of their[2Fe2S] clusters. While the molecular basis of these components remains to be determined, salient implications of these findings in terms of Qo site catalysis are discussed.},
doi = {10.1021/bi035938u},
journal = {Biochemistry (Eaton)},
issn = {0006-2960},
number = 8,
volume = 43,
place = {United States},
year = {2004},
month = {3}
}