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

Journal Article · Tue Mar 02 04:00:00 EST 2004 · Biochemistry (Eaton)

DOI:https://doi.org/10.1021/bi035938u· OSTI ID:15007033

Cooley, Jason W; Roberts, Arthur G; Bowman, Michael K; Kramer, David M; Daldal, Fevzi

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.

• Research Organization: Pacific Northwest National Lab., Richland, WA (US), Environmental Molecular Sciences Laboratory (US)
• Sponsoring Organization: US Department of Energy (US)
• DOE Contract Number: AC06-76RL01830
• OSTI ID: 15007033
• Report Number(s): PNWD-SA-6308; 2358
• Journal Information: Biochemistry (Eaton), Journal Name: Biochemistry (Eaton) Journal Issue: 8 Vol. 43; ISSN 0006-2960; ISSN BICHAW
• Country of Publication: United States
• Language: English

Related Subjects

08 HYDROGEN
ALANINES
BIOCHEMISTRY
BONDING
CATALYSIS
CHEMISTRY
CYTOCHROMES
ENVIRONMENTAL MOLECULAR SCIENCES LABORATORY
NULL
ENZYMES
HYDROGEN
IRON
MEMBRANES
MUTANTS
MUTATIONS
PROTEINS
SPECTROSCOPY
SULFUR
UBIQUINONE