Protein dynamics govern the oxyferrous state lifetime of an artificial oxygen transport protein.

It has long been known that the alteration of protein side chains that occlude or expose the heme cofactor to water can greatly affect the stability of the oxyferrous heme state. Here, we demonstrate that the rate of dynamically driven water penetration into the core of an artificial oxygen transport protein also correlates with oxyferrous state lifetime by reducing global dynamics, without altering the structure of the active site, via the simple linking of the two monomers in a homodimeric artificial oxygen transport protein using a glycine-rich loop. The tethering of these two helices does not significantly affect the active site structure, pentacoordinate heme-binding affinity, reduction potential, or gaseous ligand affinity. It does, however, significantly reduce the hydration of the protein core, as demonstrated by resonance Raman spectroscopy, backbone amide hydrogen exchange, and pKa shifts in buried histidine side chains. This further destabilizes the charge-buried entatic state and nearly triples the oxyferrous state lifetime. These data are the first direct evidence that dynamically driven water penetration is a rate-limiting step in the oxidation of these complexes. It furthermore demonstrates that structural rigidity that limits water penetration is a critical design feature in metalloenzyme construction and provides an explanation for both the failures and successes of earlier attempts to create oxygen-binding proteins.

Unwinding of the Substrate Transmembrane Helix in Intramembrane Proteolysis.

Intramembrane-cleaving proteases (I-CLiPs) activate pools of single-pass helical membrane protein signaling precursors that are key in the physiology of prokaryotic and eukaryotic cells. Proteases typically cleave peptide bonds within extended or flexible regions of their substrates, and thus the mechanism underlying the ability of I-CLiPs to hydrolyze the presumably α-helical transmembrane domain (TMD) of these membrane proteins is unclear. Using deep-ultraviolet resonance Raman spectroscopy in combination with isotopic labeling, we show that although predominantly in canonical α-helical conformation, the TMD of the established I-CLiP substrate Gurken displays 310-helical geometry. As measured by microscale thermophoresis, this substrate binds with high affinity to the I-CLiPs GlpG rhomboid and MCMJR1 presenilin homolog in detergent micelles. Binding results in deep-ultraviolet resonance Raman spectra, indicating conformational changes consistent with unwinding of the 310-helical region of the substrate's TMD. This 310-helical conformation is key for intramembrane proteolysis, as the substitution of a single proline residue in the TMD of Gurken by alanine suppresses 310-helical content in favor of α-helical geometry and abolishes cleavage without affecting binding to the I-CLiP. Complemented by molecular dynamics simulations of the TMD of Gurken, our vibrational spectroscopy data provide biophysical evidence in support of a model in which the transmembrane region of cleavable I-CLiP substrates displays local deviations in canonical α-helical conformation characterized by chain flexibility, and binding to the enzyme results in conformational changes that facilitate local unwinding of the transmembrane helix for cleavage.

Site-directed mutagenesis of the highly perturbed copper site of auracyanin D.

Type-1 copper proteins participate in redox reactions and biological catalysis. Significant variation exists within the electronic structure of type-1 copper sites, producing both blue and green proteins. Classical, "blue" sites have been extensively studied, but "green" sites have been poorly characterized. We recently discovered a green copper protein, called auracyanin D. Here, we report a series of axial ligand mutations in auracyanin D, and characterize the resulting spectral and redox changes. The resulting mutants appear blue, green, and red and vary in redox potential from +56mV to +786mV. This is the largest change in redox potential to date for any type-1 center. We found that in this green protein, modifications of the axial ligand produce significantly larger changes than similar mutations in blue type-1 copper sites.

Effects of fluidity on the ensemble structure of a membrane embedded α-helical peptide.

Melittin, the main hemolytic component of honeybee venom, is unfolded in an aqueous environment and folds into an α-helical conformation in a lipid environment. Membrane fluidity is known to affect the activity and structure of melittin. By combining two structurally sensitive optical methods, circular dichroism (CD) and deep-ultraviolet resonance Raman spectroscopy (dUVRR), we have identified distinct structural fluctuations in melittin correlated with increased and decreased 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer fluidities. CD spectra have reduced intensity at temperatures above 22°C and high concentrations of the cholesterol analog 5α-cholestan-3ß-ol indicating distortions in the α-helical structure under these conditions. No increase in the amide S is observed in the temperature-dependent dUVRR spectra, suggesting an increase in 310 -helical structure with increasing temperatures above 22°C. However, incorporation of 25 mol% 5α-cholestan-3ß-ol resulted in a small increase in the amide S intensity indicating partial unfolding of melittin.

Role of bilayer characteristics on the structural fate of aß(1-40) and aß(25-40).

The ß-amyloid (Aß) peptide is derived from the transmembrane (TM) helix of the amyloid precursor protein (APP) and has been shown to interact with membrane surfaces. To understand better the role of peptide-membrane interactions in cell death and ultimately in Alzheimer's disease, a better understanding of how membrane characteristics affect the binding, solvation, and secondary structure of Aß is needed. Employing a combination of circular dichroism and deep-UV resonance Raman spectroscopies, Aß(25-40) was found to fold spontaneously upon association with anionic lipid bilayers. The hydrophobic portion of the disease-related Aß(1-40) peptide, Aß(25-40), has often been used as a model for how its legacy TM region may behave structurally in aqueous solvents and during membrane encounters. The structure of the membrane-associated Aß(25-40) peptide was found to depend on both the hydrophobic thickness of the bilayer and the duration of incubation. Similarly, the disease-related Aß(1-40) peptide also spontaneously associates with anionic liposomes, where it initially adopts mixtures of disordered and helical structures. The partially disordered helical structures then convert to ß-sheet structures over longer time frames. ß-Sheet structure is formed prior to helical unwinding, implying a model in which ß-sheet structure, formed initially from disordered regions, prompts the unwinding and destabilization of membrane-stabilized helical structure. A model is proposed to describe the mechanism of escape of Aß(1-40) from the membrane surfaces following its formation by cleavage of APP within the membrane.

Bilayer surface association of the pHLIP peptide promotes extensive backbone desolvation and helically-constrained structures.

Despite their presence in many aspects of biology, the study of membrane proteins lags behind that of their soluble counterparts. Improving structural analysis of membrane proteins is essential. Deep-UV resonance Raman (DUVRR) spectroscopy is an emerging technique in this area and has demonstrated sensitivity to subtle structural transitions and changes in protein environment. The pH low insertion peptide (pHLIP) has three distinct structural states: disordered in an aqueous environment, partially folded and associated with a lipid membrane, and inserted into a lipid bilayer as a transmembrane helix. While the soluble and membrane-inserted forms are well characterized, the partially folded membrane-associated state has not yet been clearly described. The amide I mode, known to be sensitive to protein environment, is the same in spectra of membrane-associated and membrane-inserted pHLIP, indicating comparable levels of backbone dehydration. The amide S mode, sensitive to helical structure, indicates less helical character in the membrane-associated form compared to the membrane-inserted state, consistent with previous findings. However, the structurally sensitive amide III region is very similar in both membrane-associated and membrane-inserted pHLIP, suggesting that the membrane-associated form has a large amount of ordered structure. Where before the membrane-associated state was thought to contain mostly unordered structure and reside in a predominantly aqueous environment, we have shown that it contains a significant amount of ordered structure and rests deeper within the lipid membrane.

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.

Observation of persistent α-helical content and discrete types of backbone disorder during a molten globule to ordered peptide transition via deep-UV resonance Raman spectroscopy.

The molten globule state can aide in the folding of a protein to a functional structure and is loosely defined as an increase in structural disorder with conservation of the ensemble secondary structure content. Simultaneous observation of persistent secondary structure content with increased disorder has remained experimentally problematic. As a consequence, modeling how the molten globule state remains stable and how it facilitates proper folding remains difficult due to a lack of amenable spectroscopic techniques to characterize this class of partially unfolded proteins. Previously, deep-UV resonance Raman (dUVRR) spectroscopy has proven useful in the resolution of global and local structural fluctuations in the secondary structure of proteins. In this work, dUVRR was employed to study the molten globule to ordered transition of a model four-helix bundle protein, HP7. Both the average ensemble secondary structure and types of local disorder were monitored, without perturbation of the solvent, pH, or temperature. The molten globule to ordered transition is induced by stepwise coordination of two heme molecules. Persistent dUVRR spectral features in the amide III region at 1295-1301 and 1335-1338 cm-1 confirm previous observations that HP7 remains predominantly helical in the molten globule versus the fully ordered state. Additionally, these spectra represent the first demonstration of conserved helical content in a molten globule protein. With successive heme binding significant losses are observed in the spectral intensity of the amide III3 and S regions (1230-1260 and 1390 cm-1, respectively), which are known to be sensitive to local disorder. These observations indicate that there is a decrease in the structural populations able to explore various extended conformations, with successive heme binding events. DUVRR spectra indicate that the first heme coordination between two helical segments diminishes exploration of more elongated backbone structural conformations in the inter-helical regions. A second heme coordination by the remaining two helices further restricts protein motion.

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.

Influence of the lipid environment on valinomycin structure and cation complex formation.

Carrier-type molecular ionophores, such as the cyclic dodecadepsipeptide valinomycin, often must undergo structural changes during the binding and transport of a cation across the lipid membrane. Observing the structural fluctuations that occur during this process experimentally has proven extremely difficult due to the complexities of spectroscopic analysis of protein structure/dynamics in native lipid bilayer environments. Currently, our understanding of how valinomycin selectively transports ions across membranes is derived from atomic structures solved of the cyclic macromolecule solvated in various organic solvents and complimentary in silico dynamics experiments. We have shown recently that deep-UV excited resonance Raman spectroscopy (DUVRR) has a unique ability to characterize secondary structure content and simultaneously provide information about the relative solvation of the probed peptide backbone C.M. Halsey, J. Xiong, O. Oshokoya, J.A. Johnson, S. Shinde, J.T. Beatty, G. Ghirlanda, R.D. JiJi, J.W. Cooley, Simultaneous observation of peptide backbone lipid solvation and a-helical structure by deep-UV resonance Raman spectroscopy, ChemBioChem 12 (2011) 2125-2128, [16]. Interpretation of DUVRR spectra of valinomycin in swelled lipid and unilamellar lipid bilayer environments indicate that the uncomplexed valinomycin molecule dynamically samples both the open and closed conformations as described for the structures derived from polar and non-polar organic solvents, respectively. Upon introduction of potassium, the structure of valinomycin in swelled lipid environments resembles more closely that of the open conformation. The shift in structure upon complexation is accompanied by a significant decrease in the valinomycin DUVRR spectral amide I intensity, indicating that the open conformation is more water solubilized and is seemingly "trapped" or predominantly located close to the lipid-water interface. The trapping of the valinomycin in the act of complex of potassium at the bilayer-solvent interface and its analysis by DUVRR represents the first spectroscopic description of this state. Conversely, an opposite trend is observed in the amide I intensity upon potassium complexation in unilamellar (or extruded) vesicles, implying the predominant conformation upon potassium binding in native bilayers is one where the peptide backbone of valinomycin is desolvated as would be expected if the molecule were more readily able to traverse a bilayer interior. Interpretation of the DUVRR spectral features is also consistent with the loss or formation of hydrogen bonds observed in the open and closed structures, respectively. Valinomycin must then sample several conformations in the absence of appropriate ions depending upon its locale in the lipid bilayer until potassium causes a greater degree of closure of the open conformer and an increased residency within the more non-polar interior. The potassium induced decreased solubility enables diffusion across the membrane where potassium release can occur by equilibration at the opposite lipid water interface.

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 protein's 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.

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 bc(1) efficiently oxidizes hydroquinone to quinone, but how it performs this reaction without any leak of electrons to O(2) 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 bc(1) is critical for this process. Substitution of this residue with other amino acids decreases cytochrome bc(1) 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 O(2) by oxidative disruption of its catalytic [2Fe-2S] cluster. Hence, this Tyr residue is essential in controlling unproductive encounters between O(2) and catalytic intermediates at the quinol oxidation site of cytochrome bc(1) 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.

A structural model for across membrane coupling between the Qo and Qi active sites of cytochrome bc1.

The two spatially distant quinone-binding sites of the ubihydroquinone: cytochrome c oxidoreductase (cyt bc(1)) complex have been shown to influence one another in some fashion. This transmembrane communication alters cofactor and redox partner binding interactions and could potentially influence the timing or 'concerted' steps involved in the steady-state turnover of the homodimeric enzymes. Yet, despite several lines of evidence corroborating the coupling of the quinone binding active sites to one another, little to no testable hypothesis has been offered to explain how such a "signal" might be transmitted across the presumably rigid hydrophobic domain of the enzyme. Recently, it has been shown that this interquinone binding sites communication influences the steady-state position of the mobile [2Fe-2S] cluster containing iron sulfur protein (Sarewicz M., Dutka M., Froncisz W., Osyczka A. (2009) Biochemistry 48, 5708-5720) as mediated by at least one transmembrane helix of the b-type cyt containing subunit (Cooley, J. W., Lee, D. W., and Daldal, F. (2009) Biochemistry 48, 1988-1999). Here we provide an overview of the evidence supporting the structural coupling of these sites and provide a theoretical framework for how the redox state of a quinone at one cofactor binding site might influence the cofactor-, inhibitor-, and/or protein-protein interactions at the structurally distant opposing Q binding site.

Across membrane communication between the Q(o) and Q(i) active sites of cytochrome bc(1).

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.

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 c(y)) or a freely diffusible (cyt c2) electron carrier. Previously, we constructed a functional cyt bc1-c(y) 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-c(y) fusion complex, and used its derivatives with shorter cyt c(y) 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-c(y) 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 c(y) linkers exhibited fast, native-like ET rates to the RC via the cyt bc1. However, shortening the length of the cyt c(y) linker decreased drastically this electronic coupling between the cyt bc1-c(y) fusion complexes and the RC, thereby limiting Ps growth. The shortest and still functional cyt c(y) linker was about 45 amino acids long, showing that the minimal distance allowed between the cyt bc1-c(y) 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.

A functional hybrid between the cytochrome bc1 complex and its physiological membrane-anchored electron acceptor cytochrome cy in Rhodobacter capsulatus.

The membrane integral ubihydroquinone (QH2): cytochrome (cyt) c oxidoreductase (or the cyt bc1 complex) and its physiological electron acceptor, the membrane-anchored cytochrome cy (cyt cy), are discrete components of photosynthetic and respiratory electron transport chains of purple non-sulfur, facultative phototrophic bacteria of Rhodobacter species. In Rhodobacter capsulatus, it has been observed previously that, depending on the growth condition, absence of the cyt bc1 complex is often correlated with a similar lack of cyt cy (Jenney, F. E., et al. (1994) Biochemistry 33, 2496-2502), as if these two membrane integral components form a non-transient larger structure. To probe whether such a structural super complex can exist in photosynthetic or respiratory membranes, we attempted to genetically fuse cyt cy to the cyt bc1 complex. Here, we report successful production, and initial characterization, of a functional cyt bc1-cy fusion complex that supports photosynthetic growth of an appropriate R. capsulatus mutant strain. The three-subunit cyt bc1-cy fusion complex has an unprecedented bis-heme cyt c1-cy subunit instead of the native mono-heme cyt c1, is efficiently matured and assembled, and can sustain cyclic electron transfer in situ. The remarkable ability of R. capsulatus cells to produce a cyt bc1-cy fusion complex supports the notion that structural super complexes between photosynthetic or respiratory components occur to ensure efficient cellular energy production.

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.

Uncovering the molecular mode of action of the antimalarial drug atovaquone using a bacterial system.

Atovaquone is an antiparasitic drug that selectively inhibits electron transport through the parasite mitochondrial cytochrome bc1 complex and collapses the mitochondrial membrane potential at concentrations far lower than those at which the mammalian system is affected. Because this molecule represents a new class of antimicrobial agents, we seek a deeper understanding of its mode of action. To that end, we employed site-directed mutagenesis of a bacterial cytochrome b, combined with biophysical and biochemical measurements. A large scale domain movement involving the iron-sulfur protein subunit is required for electron transfer from cytochrome b-bound ubihydroquinone to cytochrome c1 of the cytochrome bc1 complex. Here, we show that atovaquone blocks this domain movement by locking the iron-sulfur subunit in its cytochrome b-binding conformation. Based on our malaria atovaquone resistance data, a series of cytochrome b mutants was produced that were predicted to have either enhanced or reduced sensitivity to atovaquone. Mutations altering the bacterial cytochrome b at its ef loop to more closely resemble Plasmodium cytochrome b increased the sensitivity of the cytochrome bc1 complex to atovaquone. A mutation within the ef loop that is associated with resistant malaria parasites rendered the complex resistant to atovaquone, thereby providing direct proof that the mutation causes atovaquone resistance. This mutation resulted in a 10-fold reduction in the in vitro activity of the cytochrome bc1 complex, suggesting that it may exert a cost on efficiency of the cytochrome bc1 complex.

The raised midpoint potential of the [2Fe2S] cluster of cytochrome bc1 is mediated by both the Qo site occupants and the head domain position of the Fe-S protein subunit.

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.