The Raised Midpoint Potential of the [2Fe2S] Cluster of Cytochromebc₁Is Mediated by Both the Q_oSite 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 bc(1) complex have increased redox midpoint potentials (E(m)) for their [2Fe2S] clusters. The alteration of the E(m) in these strains, which 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 (Q(o)) site [Darrouzet, E., Valkova-Valchanova, M., and Daldal, F. (2002) J. Biol. Chem. 277, 3464-3470]. Subsequently, the E(m) changes have been proposed to be predominantly due to a stronger or more stabilized hydrogen bonding between the reduced [2Fe2S] cluster and the Q(o) 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 electron paramagnetic resonance (EPR) spectroscopy and the findings indicated that the wild type and mutant proteins interactions with Q are similar. Moreover, when the Q(pool) was chemically depleted, the E(m) of the [2Fe2S] cluster in mutant bc(1) complexes remained more positive than a similarly treated native enzyme (e.g., the [2Fe2S] E(m) of the +2Ala mutant was 55 mV more positive than the wild type). These data suggest that the increased E(m) of the [2Fe2S] cluster in the +nAla mutants is in part due to the cluster's 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 Q(o) 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, Q(pool) 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 the +2Ala mutant than the Q-depleted membranes of a wild-type strain. Therefore, additional component(s) that are independent of Q(o) 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 Q(o) site catalysis are discussed.

Binding Dynamics at the Quinone Reduction (Qi) Site Influence the Equilibrium Interactions of the Iron Sulfur Protein and Hydroquinone Oxidation (Qo) Site of the Cytochromebc1Complex

“…Addition of antimycin A to ordered membrane samples alters the EPR spectral shape of the [2Fe-2S] cluster of the cyt bc 1 In contrast to what was observed with the cyt b H and b L hemes, addition of either antimycin A or HQNO to membranes exhibited no discernable effect on the EPR transitions of the [2Fe-2S] cluster in non-ordered (or powder) sample EPR spectra ( Figures 3 and 4), as has been described earlier (26). Unlike the tensor averaged powder sample EPR spectra, use of ordered membrane samples increases both the spectral resolution of a given transition in the EPR spectrum, and also yields specific information about the relative orientation (and the relative numbers of different orientations) of a metal cluster in a given sample.…”

“…In these cases, the total amount of the Fe/S protein residing at the cyt b surface was diminished, and the EPR g y transition of the [2Fe-2S] cluster shifted to slightly lower magnetic field positions. Furthermore, in similarly ordered membranes increased mobility of the Fe/S protein was also accompanied by a diminished orientation dependence of the [2Fe-2S] cluster spectrum (26). The similarities of these observations to the data obtained by addition of antimycin A suggested that the membrane embedded cyt bc 1 has an increased mobility of the Fe/S protein subunit.…”

“…In the case of the H217R mutant, untreated membrane samples already had a [2Fe-2S] cluster EPR line shape lacking a clear g x = 1.8 transition and a somewhat decreased orientation dependence. When treated with antimycin A they looked similar to those obtained using the soluble portion of the Fe/S protein (38), or to the ordered samples containing native cyt bc 1 either treated with myxothiazol or chemically Q depleted (26). Conversely, the orientation dependence of these spectra was still visible with samples treated with HQNO.…”

“…Ordered membrane sample preparation was modified from those outlined in (24) and (25) and described in detail in (26). Chemical depletion of Q from chromatophore membranes (< 1 Q per reaction center) was carried out as outlined in (27).…”

X-Ray Structure of Rhodobacter Capsulatus Cytochrome bc1: Comparison with its Mitochondrial and Chloroplast Counterparts

“…The same crystal form has been obtained without stigmatellin, however the resolution (7 Å) was not high enough to describe the occupation of the Qo site. Efforts are currently under way to crystallize one of the "neck" mutants (Darrouzet et al 2000;Cooley et al 2004), which seem to hold the ISP prefferentially in a position similar to the stigmatellin position, in the absence of stigmatellin.…”