Exploration of Ligands to the Qi Site Semiquinone in the bc 1 Complex Using High-resolution EPR

Abstract

Pulsed EPR spectroscopy was used to explore the structural neighborhood of the semiquinone (SQ) stabilized at the Q i site of the bc 1 complex of Rhodobacter sphaeroides (EC 1.10.2.2) and to demonstrate that the nitrogen atom of a histidine imidazole group donates an H-bond to the SQ. Crystallographic structures show two different configurations for the binding of ubiquinone at the Q i site of mitochondrial bc 1 complexes in which histidine (His-201 in bovine sequence) is either a direct H-bond donor or separated by a bridging water. The paramagnetic properties of the SQ formed at the site provide an independent method for studying the liganding of this intermediate species. The antimycin-sensitive SQ formed at the Q i site by either equilibrium redox titration, reduction of the oxidized complex by ascorbate, or addition of decylubihydroquinone to the oxidized complex in the presence of myxothiazol all showed similar properties. The electron spin echo envelope modulation spectra in the 14 N region were dominated by lines with frequencies at 1.7 and 3.1 MHz. Hyperfine sublevel correlation spectroscopy spectra showed that these were contributed by a single nitrogen. Further analysis showed that the 14 N nucleus was characterized by an isotropic hyperfine coupling of ϳ0.8 MHz and a quadrupole coupling constant of ϳ0.35 MHz. The nitrogen was identified as the N-⑀ or N-␦ imidazole nitrogen of a histidine (it is likely to be His-217, or His-201 in bovine sequence). A distance of 2.5-3.1 Å for the O-N distance between the carbonyl of SQ and the nitrogen was estimated. The mechanistic significance is discussed in the context of a dynamic role for the movement of His-217 in proton transfer to the site.The bc 1 complex family of enzymes plays a central role in all the main pathways of energy conversion, being directly responsible for ϳ30% of all the energy transduction of the biosphere (1-3). The complexes in Rhodobacter exemplify the simplest of these enzymes, with only three or four subunits, including the highly conserved catalytic core common to bacterial and mitochondrial complexes. It is generally accepted that the complex operates through a protonmotive Q cycle (2, 4, 5). Three catalytic subunits, cytochrome b, cytochrome c 1 , and the Rieske iron-sulfur protein, house the mechanism. Two separate internal electron transfer chains connect three catalytic sites for external substrates. At one site, cytochrome c 1 is oxidized by cytochrome c 2 . Two catalytic sites in cytochrome b are involved in the oxidation or reduction of ubiquinone. At the quinol oxidizing site, one electron from quinol is passed to the ironsulfur protein, which transfers it to cytochrome c 1 , whereas the semiquinone (SQ) 1 produced is oxidized by another chain consisting of the two b-hemes of cytochrome b in the bifurcated reaction. At the quinone-reducing site (Q i site), electrons from the b-heme chain are used to generate quinol. The integration of the oxidation and reduction reactions with the release or uptake of protons in the aqueous pha...

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

“…We found no electron spin echo envelope modulation evidence for a hyperfine coupling between the new SQ species and N-nuclei as might be expected if the SQ was H-bonded to an amide or histidine nitrogen with accompanying SQ spin density on the N (37,52). Although these couplings have often been difficult to detect, the N-modulation depth in our system appears significantly weaker than for SQs in some related systems, e.g., Q A in photosystem II (e.g., ref.…”

“…In this experiment AA blocked the Q i site, thus eliminating signals from any stabilized SQ at this site, while preventing reoxidation of the cyt b chain (and partially oxidized Q o site intermediates) through the Q i site. The free radical signal exhibited a g-factor of Ϸ2.0054 and a line width (11.9 gauss) distinct from that of the narrower Q i site SQ signal (Ϸ8.0 gauss) (37).…”

Resolving the EPR Spectra in the Cytochrome bc 1 Complex from Saccharomyces cerevisiae

“…More recently similar experiments have been performed on the bacterial bc 1 complex from Rhodobacter (Rb.) sphaeroides and a direct binding to histidine has been proposed [23,24]. When applying EPR spectroscopy to study electron-transfer intermediates in mitochondrial or bacterial respiratory chains, it is important to realize that presence of several paramagnetic species is a common occurrence.…”

Simultaneous reduction of iron–sulfur protein and cytochrome b L during ubiquinol oxidation in cytochrome bc 1 complex

“…Then the second electron of ubiquinol is passed through the ''low-potential chain'' consisting of cytochromes b L and b H to reduce ubiquinone or ubisemiquinone bound at the Qn site. Although crystallographic data clearly indicates the presence of two separated quinone binding domains: one for ubiquinol oxidation (Qp) and the other for quinone reduction (Qn), only the binding of ubiquinone (17)(18)(19) and the presence of ubisemiquinone radical (20)(21)(22) at the Qn site were demonstrated. The binding of ubiquinol or ubiquinone at the Qp site has not been detected even though the binding of various Qp site inhibitors (18,23,24) is well known.…”