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

Abstract

Multiple instances of low potential electron transport pathway inhibitors that affect the structure of the cytochrome (cyt) bc 1 complex to varying degrees, ranging from changes in hydroquinone (QH 2 ) oxidation and cyt c 1 reduction kinetics, to proteolytic accessibility of the hinge region of the iron-sulfur containing subunit (Fe/S protein), have been reported. However, no instance has been documented of any ensuing change on the environment(s) of the [2Fe-2S] cluster. In this work, this issue was addressed in detail by taking advantage of the increased spectral and spatial resolution obtainable with orientation dependent electron paramagnetic resonance (EPR) spectroscopic analysis of ordered membrane preparations. For the first time, perturbation of the low potential electron transport pathway by Q i site inhibitors or various mutations was shown to change the EPR spectra of both the cyt b hemes and the [2Fe-2S] cluster of the Fe/S protein. In particular, two interlinked effects of Q i site modifications on the Fe/S subunit, one changing the local environment of its [2Fe-2S] cluster, and a second affecting the mobility of this subunit are revealed. Remarkably, different inhibitors and mutations at or near the Q i site induce these two effects differently, indicating that the events occurring at the Q i site affect the global structure of the cyt bc 1. Furthermore, occupancy of discrete Q i site subdomains differently impede the location of the Fe/S protein at the Q o site. These findings led us to propose that antimycin A and HQNO mimic the presence of QH 2 and Q at the Q i site, respectively. Implications of these findings in respect to the Q o -Q i sites communications and to multiple turnovers of the cyt bc 1 are discussed. KeywordsAntimycin A; cytochrome bc 1 ; complex III; Rhodobacter capsulatus; photosynthesis and respiration; energy transduction ABBREVIATIONSElectron paramagnetic resonance (EPR); [2Fe-2S] cluster containing protein (Fe/S); quinone (Q); cytochrome b (cyt b); hydroquinone (QH 2 ); hydroquinone:cytochrome (cyt) c oxidoreductase (cyt bc 1 ); 2-heptyl-4-hydroxyquinoline N-oxide (HQNO); 2-nonyl-4-hydroxyquinoline N-oxide (NQNO); quinone reduction site (Q i ); hydroquinone oxidation site (Q o ) *To whom correspondence should be addressed: Phone: (215) 898-4394 Fax: (215) 898-8780 E-mail:fdaldal@sas.upenn.edu. This work was supported by NIH grant R01 GM 38237 to F. D. and NIH F32 GM 65791 and AHA #0425515U fellowships to J. W. C. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2006 February 2. Published in final edited form as:Biochemistry. 2005 August 9; 44(31): 10520-10532. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe hydroquinone (QH 2 ):cytochrome (cyt) c oxidoreductase (cyt bc 1 ) is an essential component of the mitochondrial and most bacterial respiratory electron transport pathways (1). A sister complex, the cyt b 6 f, is also a part of the photosynthetic electron transport chains of the chloroplasts of highe...

A semiquinone intermediate generated at the Q o site of the cytochrome bc 1 complex: Importance for the Q-cycle and superoxide production

“…In some models the SQ is thermodynamically stabilized (45), thus lowering its reactivity with O 2 ; in other models it is specifically destabilized (3,36), limiting its reactivity with O 2 by lowering its steady-state concentration. Some models posit that the SQ is shielded from O 2 within the Q o site (3), while access to the site or its reactivity is proposed to be allosterically (46,47) or electrostatically (14,27) gated. Still, other models deny the existence of an SQ intermediate altogether (21).…”

Similar Transition States Mediate the Q-cycle and Superoxide Production by the Cytochrome bc1 Complex

“…Several groups have suggested reasonable models that entail conformational or electrostatic gating of the Q o site reactions (52)(53)(54)(55)(56)(57). In these models, the activity of the Q o site is gated by the properties of other components of the complex.…”

“…In these models, the activity of the Q o site is gated by the properties of other components of the complex. For example, the Q o site might not be able to bind substrate or becomes redox-inactive when cyt b L is reduced (28), the Q i site is altered (54), substrate occupancy in one-half of the operational dimer (50) may inhibit activity in the other half of the dimer (53). Under normal conditions, such gating could effectively prevent side reactions but might be susceptible to slippage over large time scales, leading to the observed differences in SO production rates in the uninhibited and inhibited cases.…”

Electronic Connection Between the Quinone and CytochromecRedox Pools and Its Role in Regulation of Mitochondrial Electron Transport and Redox Signaling

“…Although the crystal structures do not show any significant structural changes caused by binding of antimycin (126), a potent inhibitor of the Q i site, biochemical and spectroscopic studies have implicated that it might influence the average position of FeS head domain in the complex (51,222,260). While this long-range structural effect has been exploited by various models that propose allostery within the dimer of cytochrome bc 1 (50,(52)(53)(54), the origin and the time domain of this inhibitorinduced effect are unknown.…”