Ultrafast photochemistry of the bc₁complex

Abstract

We present a full investigation of ultrafast light-induced events in the membraneous cytochrome bc1 complex by transient absorption spectroscopy. This energy-transducing complex harbors four redox-active components per monomer: heme c1, two 6-coordinate b-hemes and a [2Fe-2S] cluster. Using excitation of these components in different ratios under various excitation conditions, probing in the full visible range and under three well-defined redox conditions, we demonstrate that for all ferrous hemes of the complex photodissociation of axial ligands takes place and that they rebind in 5–7 ps, as in other 6-coordinate heme proteins, including cytoglobin, which is included as a reference in this study. By contrast, the signals are not consistent with photooxidation of the b hemes. This conclusion contrasts with a recent assessment based on a more limited data set. The binding kinetics of internal and external ligands are indicative of a rigid heme environment, consistent with the electron transfer function. We also report, for the first time, photoactivity of the very weakly absorbing iron-sulfur center. This yields the unexpected perspective of studying photochemistry, initiated by excitation of iron-sulfur clusters, in a range of protein complexes.

Fluorescent iron‑sulfur centers: Photochemistry of the PetA Rieske protein from Aquifex aeolicus

“…Indeed, the number of Fe-S proteins [8,9]. Also, we have previously suggested photo-oxidation of the Rieske 2Fe-2S cluster in the Rhodobacter capsulatus bc1 complex, but this assessment was complicated by the dominant absorption of the hemes in this complex [7]. Altogether, our present work demonstrates such photo-oxidation and moreover shows that in the same protein photoreduction and photooxidation of Fe-S centers can take place.…”

“…The initial processes following the absorption of light by such systems have been investigated to some extent only in very recent years. In 2017, as an unexpected side result of a transient absorption study on the photochemistry of hemes in a bacterial cytochrome bc1 complex that takes place on the timescale of a few picoseconds, we reported a weak signal lasting much longer and occurring upon excitation in the blue spectral region [7]. This signal was assigned to photo-oxidation of reduced [2Fe-2S] centers.…”

“…Following precursory indications of involvement of long-lived photoproducts assignable to the Rieske 2Fe-2S protein moiety of the intact bc1 complex of a photosynthetic bacterium [7], we have now presented comprehensive spectroscopic data on the initial lightinduced processes in a Rieske 2Fe-2S protein itself. Isolated Rieske proteins are notoriously unstable, but soluble PetA from the hyperthermophilic bacterium A. aeolicus could be prepared and maintained in both the oxidized and reduced form for sufficiently long times to allow detailed spectroscopic investigations.…”

“…In the present work, we investigate the excited state and product state dynamics on the femtosecond to nanosecond timescale of a Rieske [2Fe-2S] protein [13,14]. This choice was inspired by our previous work on the respiratory cytochrome bc1 complex from the purple bacterium Rhodobacter capsulatus [7]. Rieske proteins are essential constituents of cytochrome bc1 complexes, with their [2Fe-2S] cluster acting as an electron transfer intermediate between external quinones and the c1 heme.…”

Ultrafast Dynamics and Vibrational Relaxation in Six-Coordinate Heme Proteins Revealed by Femtosecond Stimulated Raman Spectroscopy

“…In the literature, the photolysis of amino acid residues, i.e., histidine and methionine, from ferrous hemes has been mainly investigated with TA spectroscopy. ,,,− The only Raman evidence of bond breaking, obtained with a picosecond time resolution by spontaneous TR 3 , is the appearance in cyt c of the iron–histidine band, following vibrational coherence spectroscopy assignments . Vibrational cooling subsequent to photolysis has been studied on the picosecond time scale by TR 3 , but here the poor spectral and temporal resolution limit the obtainable information on the evolution of the dissociated configuration and in particular does not allow us to resolve hot bands.…”