Jason W. Cooley

Binding Dynamics at the Quinone Reduction (Qi) Site Influence the Equilibrium Interactions of the Iron Sulfur Protein and Hydroquinone Oxidation (Qo) Site of the Cytochrome bc1 Complex

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.

The Cytochrome bc1 Complex and its Homologue the b6f Complex: Similarities and Differences

The ubihydroquinone:cytochrome c oxidoreductase (also called complex III, or bc1 complex), is a multi subunit enzyme encountered in a very broad variety of organisms including uni- and multi-cellular eukaryotes, plants (in their mitochondria) and bacteria. Most bacteria and mitochondria harbor various forms of the bc1 complex, while plant and algal chloroplasts as well as cyanobacteria contain a homologous protein complex called plastohydroquinone:plastocyanin oxidoreductase or b6f complex. Together, these enzyme complexes constitute the superfamily of the bc complexes. Depending on the physiology of the organisms, they often play critical roles in respiratory and photosynthetic electron transfer events, and always contribute to the generation of the proton motive force subsequently used by the ATP synthase. Primarily, this review is focused on comparing the ‘mitochondrial-type’ bc1 complex and the ‘chloroplast-type’ b6f complex both in terms of structure and function. Specifically, subunit composition, cofactor content and assembly, inhibitor sensitivity, proton pumping, concerted electron transfer and Fe—S subunit large-scale domain movement of these complexes are discussed. This is a timely undertaking in light of the structural information that is emerging for the b6f complex.

Across Membrane Communication between the Qo and Qi Active Sites of Cytochrome bc1

The ubihydroquinone:cytochrome c oxidoreductase (cyt bc(1)) contains two catalytically active domains, termed the hydroquinone oxidation (Q(o)) and quinone reduction (Q(i)) sites, which are distant from each other by over 30 A. Previously, we have reported that binding of inhibitors to the Q(i) site on one (n) side of the energy-transducing membrane changes the local environment of the iron-sulfur (Fe/S) protein subunit residing in the Q(o) site on the other (p) side of the lipid bilayer [Cooley, J. W., Ohnishi, T., and Daldal, F. (2005) Biochemistry 44, 10520-10532]. These findings best fit a model whereby the Q(o) and Q(i) sites of the cyt bc(1) are actively coupled in spite of their distant locations. Because the Fe/S protein of the cyt bc(1) undergoes a large-scale (macro) domain movement during catalysis, we examined various macromobility-defective Fe/S subunit mutants to assess the role of this motion on the coupling of the active sites and also during the multiple turnovers of the enzyme. By monitoring the changing environments of the Fe/S protein [2Fe-2S] cluster upon addition of Q(i) site inhibitors in selected mutants, we found that the Q(o)-Q(i) site interactions manifest differently depending on the ability of the Fe/S protein to move between the cytochrome b and cytochrome c(1) subunits of the enzyme. In the presence of antimycin A, an immobile Fe/S protein mutant exhibited no changes in its EPR spectra. In contrast, mobility-restricted mutants showed striking alterations in the EPR line shapes and revealed two discrete subpopulations in respect to the [2Fe-2S] cluster environments at the Q(o) site. These findings led us to conclude that the mobility of the Fe/S protein is involved in its response to the occupancy of the Q(i) site by different molecules. We propose that the heterogeneity seen might reflect the distinct responses of the two Fe/S proteins at the Q(o) sites of the dimeric enzyme upon the occupancy of the Q(i) sites and discuss it in terms of the function of the dimeric cyt bc(1) during its multiple turnovers.

The Cytochrome bc 1 and Related bc Complexes: The Rieske/Cytochrome b Complex as the Functional Core of a Central Electron/Proton Transfer Complex

The cytochrome (Cyt) bc 1 and related complexes play a central role in purple bacterial photosynthesis, transferring electrons between electron carriers reduced and oxidized by the photochemical reaction centers, oxidizing quinol (QH2) and reducing Cyt c while translocating protons via some variation of the Q-cycle mechanism. In this chapter, we discuss recent advances in the biochemical, biophysical and evolutionary understanding of these complexes. The mechanistic core of these complexes, conserved over billions of years, contains the Cyt b protein (with its two associated b-type hemes) and the Rieske iron-sulfur center. Together, this central core performs the central (and well-conserved) reaction of the Q-cycle, that is the ‘bifurcated’ oxidation of QH2 at the quinol oxidation (Qo) site, with two electrons sent to different acceptors, one to the Rieske iron-sulfur center and the other to the Cyt b chain. The subsequent reactions of the Q-cycle, involving the reduction of secondary carriers (a high potential Cyt c in the case of purple bacteria) and quinone at the quinone reduction (Qi) site are less well conserved both in terms of structure and mechanism. We thus use the term Rieske/Cytochrome b (RB) complexes for these enzymes. Key issues surrounding the mechanisms of the RB complexes are discussed, including a series of currently debated models for the avoidance of deleterious side reactions within the Qo site, the mechanism of stabilization of semiquinone intermediates within the Qi site, and the role of the pivoting iron-sulfur protein subunit.

Loss of a Conserved Tyrosine Residue of Cytochrome b Induces Reactive Oxygen Species Production by Cytochrome bc1

Production of reactive oxygen species (ROS) induces oxidative damages, decreases cellular energy conversion efficiencies, and induces metabolic diseases in humans. During respiration, cytochrome bc1 efficiently oxidizes hydroquinone to quinone, but how it performs this reaction without any leak of electrons to O2 to yield ROS is not understood. Using the bacterial enzyme, here we show that a conserved Tyr residue of the cytochrome b subunit of cytochrome bc1 is critical for this process. Substitution of this residue with other amino acids decreases cytochrome bc1 activity and enhances ROS production. Moreover, the Tyr to Cys mutation cross-links together the cytochrome b and iron-sulfur subunits and renders the bacterial enzyme sensitive to O2 by oxidative disruption of its catalytic [2Fe-2S] cluster. Hence, this Tyr residue is essential in controlling unproductive encounters between O2 and catalytic intermediates at the quinol oxidation site of cytochrome bc1 to prevent ROS generation. Remarkably, the same Tyr to Cys mutation is encountered in humans with mitochondrial disorders and in Plasmodium species that are resistant to the anti-malarial drug atovaquone. These findings illustrate the harmful consequences of this mutation in human diseases.

Cytochrome bc1-cy Fusion Complexes Reveal the Distance Constraints for Functional Electron Transfer Between Photosynthesis Components

Photosynthetic (Ps) growth of purple non-sulfur bacteria such as Rhodobacter capsulatus depends on the cyclic electron transfer (ET) between the ubihydroquinone (QH2): cytochrome (cyt) c oxidoreductases (cyt bc1 complex), and the photochemical reaction centers (RC), mediated by either a membrane-bound (cyt cy) or a freely diffusible (cyt c2) electron carrier. Previously, we constructed a functional cyt bc1-cy fusion complex that supported Ps growth solely relying on membrane-confined ET ( Lee, D.-W., Ozturk, Y., Mamedova, A., Osyczka, A., Cooley, J. W., and Daldal, F. (2006) Biochim. Biophys. Acta 1757, 346-352 ). In this work, we further characterized this cyt bc1-cy fusion complex, and used its derivatives with shorter cyt cy linkers as “molecular rulers” to probe the distances separating the Ps components. Comparison of the physicochemical properties of both membrane-embedded and purified cyt bc1-cy fusion complexes established that these enzymes were matured and assembled properly. Light-activated, time-resolved kinetic spectroscopy analyses revealed that their variants with shorter cyt cy linkers exhibited fast, native-like ET rates to the RC via the cyt bc1. However, shortening the length of the cyt cy linker decreased drastically this electronic coupling between the cyt bc1-cy fusion complexes and the RC, thereby limiting Ps growth. The shortest and still functional cyt cy linker was about 45 amino acids long, showing that the minimal distance allowed between the cyt bc1-cy fusion complexes and the RC and their surrounding light harvesting proteins was very short. These findings support the notion that membrane-bound Ps components form large, active structural complexes that are “hardwired” for cyclic ET.

Simultaneous observation of peptide backbone lipid solvation and α-helical structure by deep-UV resonance Raman spectroscopy.

Despite a variety of methodologies aimed at improving membrane protein structure analysis, information about these proteins in their native membrane environments remains scarce. Currently, no structurally sensitive spectroscopic techniques are capable of co-determining ensemble structural content and localized lipid versus aqueous solvation information. Here, we describe the first deep-UV (lex<210 nm) resonance Raman (dUVRR) spectra of a model a-helical peptide embedded in a membrane-mimetic environment, confirming sensitivity to secondary structure content and revealing sensitivity of dUVRR to the lipid solvation of the peptide backbone. Analyses of membrane protein structural dynamics are hampered by the experimental difficulties associated with elucidating structural changes and correlating those changes to their respective solvation by the nonpolar lipid or surfactant versus the aqueous phases. No kinetically amenable spectroscopic techniques are capable of delineating subtle changes in protein structure while simultaneously reporting on that structure’s solvation without protein modification by deuterium exchange, isotope labeling, mutagenesis or post-translational spin/fluorophore labeling. Glimpses of the dynamics and stabilizing forces involved with protein folding and insertion into membranes have recently been gleaned by UV excited resonance Raman spectroscopy focused on excitation wavelengths specific for aromatic residues (lex>220 nm). Deep-UV (lex< 210 nm) excitation, which has been a valuable tool for analyzing the structure of soluble proteins by accessing the p!p* transition of the peptide backbone vibrational modes and their dynamics, has not been previously explored successfully for this class of hydrophobic proteins. The dUVRR protein spectral response consists of four peptide backbone related amide (Am) responses—I (C=O stretching), II (in phase C-H/N-H stretching/bending), III (out of phase C-H/N-H stretching/bending) and S (coupled C-H/N-H bending; alternately referred to as CaHb). [2a] The combinations of Am mode positions and intensities are strongly correlated to the constraints imparted by particular secondary structures with soluble proteins. Solvent interaction and its extent with the peptide backbone can also influence the Am mode spectral positions in dUVRR and IR and intensities in dUVRR alone. Theoretical calculations with Nmethylacetamide (NMA) in different solvent polarities have revealed that the solvent-dependent Am I intensity differences seen in the dUVRR spectra, but not the IR spectra are derived from the sensitivity of the former technique to the polarizability term of the C=O bond. Herein, we present evidence that a surfactant-solubilized protein region also has altered Am mode intensities, especially in the C=O stretching region. As a model for the common a-helical membrane-embedded protein domain, we have examined the de novo designed ME1 peptide, which contains a single hydrophobic a-helical segment encompassing roughly 75 % of the total peptide backbone. Like its parent protein, it is extremely insoluble in aqueous solvents and only forms stable a-helical homodimers within a micellar environment. The dUVRR spectrum using an excitation source of 197 nm of a dodecyl phosphocholine (DPC)-solubilized ME1 sample contains aromatic side chain-derived modes (1180–1210 and 1580–1620 cm ) arising from the single tyrosine and phenylalanine residues within the peptide sequence (Figure 1). Peptide backbone contributions can also be assigned for the Am I (1658 cm ), II (1546 cm ) and III (1260–1340 cm ) modes and a smaller feature where the Am S (1400 cm ) mode would be expected. The Am III mode’s position, coupled to the limited extent of the Am S contribu-

Deep-UV resonance Raman analysis of the Rhodobacter capsulatus cytochrome bc₁complex reveals a potential marker for the transmembrane peptide backbone.

Classical strategies for structure analysis of proteins interacting with a lipid phase typically correlate ensemble secondary structure content measurements with changes in the spectroscopic responses of localized aromatic residues or reporter molecules to map regional solvent environments. Deep-UV resonance Raman (DUVRR) spectroscopy probes the vibrational modes of the peptide backbone itself, is very sensitive to the ensemble secondary structures of a protein, and has been shown to be sensitive to the extent of solvent interaction with the peptide backbone [ Wang , Y. , Purrello , R. , Georgiou , S. , and Spiro , T. G. ( 1991 ) J. Am. Chem. Soc. 113 , 6368 - 6377 ]. Here we show that a large detergent solubilized membrane protein, the Rhodobacter capsulatus cytochrome bc(1) complex, has a distinct DUVRR spectrum versus that of an aqueous soluble protein with similar overall secondary structure content. Cross-section calculations of the amide vibrational modes indicate that the peptide backbone carbonyl stretching modes differ dramatically between these two proteins. Deuterium exchange experiments probing solvent accessibility confirm that the contribution of the backbone vibrational mode differences are derived from the lipid solubilized or transmembrane α-helical portion of the protein complex. These findings indicate that DUVRR is sensitive to both the hydration status of a proteins peptide backbone, regardless of primary sequence, and its secondary structure content. Therefore, DUVRR may be capable of simultaneously measuring protein dynamics and relative water/lipid solvation of the protein.

Protein conformational changes involved in the cytochrome bc1 complex catalytic cycle.

Early structures of the cytochrome bc1 complex revealed heterogeneity in the position of the soluble portion of the Rieske iron sulfur protein subunit, implicating a movement of this domain during function. Subsequent biochemical and biophysical works have firmly established that the motion of this subunit acts in the capacity of a conformationally assisted electron transfer step during the already complicated catalytic mechanism described within the modified version of Peter Mitchells Q cycle. How the movement of this subunit is initiated or how the frequency of its motion is controlled as a function of other steps during the catalysis remain topics of debate within the active research communities. This review addresses the historical aspects of the discovery and description of this movement, while attempting to provide a context for the involvement of conformational motion in the catalysis and efficiency of the enzyme. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

Metalloproteins diversified: the auracyanins are a family of cupredoxins that stretch the spectral and redox limits of blue copper proteins.

The metal sites of electron transfer proteins are tuned for function. The type 1 copper site is one of the most utilized metal sites in electron transfer reactions. This site can be tuned by the protein environment from +80 mV to +680 mV in typical type 1 sites. Accompanying this huge variation in midpoint potentials are large changes in electronic structure, resulting in proteins that are blue, green, or even red. Here, we report a family of blue copper proteins, the auracyanins, from the filamentous anoxygenic phototroph Chloroflexus aurantiacus that display the entire known spectral and redox variations known in the type 1 copper site. C. aurantiacus encodes four auracyanins, labeled A-D. The midpoint potentials vary from +83 mV (auracyanin D) to +423 mV (auracyanin C). The electronic structures vary from classical blue copper UV-vis absorption spectra (auracyanin B) to highly perturbed spectra (auracyanins C and D). The spectrum of auracyanin C is temperature-dependent. The expansion and divergent nature of the auracyanins is a previously unseen phenomenon.