Detergents as tools in membrane biochemistry.

Detergents are invaluable tools for studying membrane proteins. However, these deceptively simple, amphipathic molecules exhibit complex behavior when they self-associate and interact with other molecules. The phase behavior and assembled structures of detergents are markedly influenced not only by their unique chemical and physical properties but also by concentration, ionic conditions, and the presence of other lipids and proteins. In this minireview, we discuss the various aggregate forms detergents assume and some misconceptions about their structure. The distinction between detergents and the membrane lipids that they may (or may not) replace is emphasized in the most recent high resolution structures of membrane proteins. Detergents are clearly friends and foes, but with the knowledge of how they work, we can use the increasing variety of detergents to our advantage.

Fast deuterium access to the buried magnesium/manganese site in cytochrome c oxidase.

The recently determined crystal structures of bacterial and bovine cytochrome c oxidases show an area of organized water within the protein immediately above the active site where oxygen chemistry occurs. A pathway for exit of protons or water produced during turnover is suggested by possible connections of this aqueous region to the exterior surface. A non-redox-active Mg(2+) site is located in the interior of this region, and our previous studies [Florens, L., Hoganson, C., McCracken, J., Fetter, J., Mills, D., Babcock, G. T., and Ferguson-Miller, S. (1998) in Phototropic Prokaryotes (Peschek, G. A., Loeffelhard, W., and Schmetterer, G., Eds.) Kluwer Academic/Plenum, New York] have shown that the protons of water molecules that coordinate the metal can be exchanged within minutes of mixing with (2)H(2)O. Here we examine the extent and rate of deuterium exchange, using a combination of rapid freeze-quench and electron spin echo envelope modulation (ESEEM) analysis of Mn(2+)-substituted cytochrome c oxidase, which retains full activity. In the oxidized enzyme at room temperature, deuterium exchange at the Mn(2+) site occurs in less than 11 ms, which corresponds to an apparent rate constant higher than 3000 s(-1). The extent of deuterium substitution is dependent on the concentration of (2)H(2)O in the sample, indicative of rapid equilibrium, with three inner sphere (2)H(2)O exchanged per Mn(2+). This indicates that the water ligands of the Mn(2+)/Mg(2+) site, or the protons of these waters, can exchange with bulk solvent at a rate consistent with a role for this region in product release during turnover.

C-terminal truncation and histidine-tagging of cytochrome c oxidase subunit II reveals the native processing site, shows involvement of the C-terminus in cytochrome c binding, and improves the assay for proton pumping.

To enable metal affinity purification of cytochrome c oxidase reconstituted into phospholipid vesicles, a histidine-tag was engineered onto the C-terminal end of the Rhodobacter sphaeroides cytochrome c oxidase subunit II. Characterization of the natively processed wildtype oxidase and artificially processed forms (truncated with and without a his-tag) reveals Km values for cytochrome c that are 6-14-fold higher for the truncated and his-tagged forms than for the wildtype. This lowered ability to bind cytochrome c indicates a previously undetected role for the C-terminus in cytochrome c binding and is mimicked by reduced affinity for an FPLC anion exchange column. The elution profiles and kinetics indicate that the removal of 16 amino acids from the C-terminus, predicted from the known processing site of the Paracoccus denitrificans oxidase, does not produce the same enzyme as the native processing reaction. MALDI-TOF MS data show the true C-terminus of subunit II is at serine 290, three amino acids longer than expected. When the his-tagged form is reconstituted into lipid vesicles and further purified by metal affinity chromatography, significant improvement is observed in proton pumping analysis by the stopped-flow method. The improved kinetic results are attributed to a homogeneous, correctly oriented vesicle population with higher activity and less buffering from extraneous lipids.

Redesign of the proton-pumping machinery of cytochrome c oxidase: proton pumping does not require Glu(I-286).

One of the putative proton-transfer pathways leading from solution toward the binuclear center in many cytochrome c oxidases is the D-pathway, so-called because it starts with a highly conserved aspartate [D(I-132)] residue. Another highly conserved amino acid residue in this pathway, glutamate(I-286), has been indicated to play a central role in the proton-pumping machinery of mitochondrial-type enzymes, a role that requires a movement of the side chain between two distinct positions. In the present work we have relocated the glutamate to the opposite side of the proton-transfer pathway by constructing the double mutant EA(I-286)/IE(I-112). This places the side chain in about the same position in space as in the original enzyme, but does not allow for the same type of movement. The results show that the introduction of the second-site mutation, IE(I-112), in the EA(I-286) mutant enzyme results in an increase of the enzyme activity by a factor of >10. In addition, the double mutant enzyme pumps approximately 0.4 proton per electron. This observation restricts the number of possible mechanisms for the operation of the redox-driven proton pump. The proton-pumping machinery evidently does require the presence of a protonatable/polar residue at a specific location in space, presumably to stabilize an intact water chain. However, this residue does not necessarily have to be at a strictly conserved location in the amino acid sequence. In addition, the results indicate that E(I-286) is not the "proton gate" of cytochrome c oxidase controlling the flow of pumped protons from one to the other side of the membrane.

Where is 'outside' in cytochrome c oxidase and how and when do protons get there?

Cytochrome c oxidase moves both electrons and protons in its dual role as a terminal electron acceptor and a contributor to the proton motive force which drives the formation of ATP. Although the sequence of electron transfer events is well-defined, the correlated mechanism and routes by which protons are translocated across the membrane are not. A recent model [Michel, Proc. Natl. Acad. Sci. USA 95 (1998) 12819] offers a detailed molecular description of when and how protons are translocated through the protein to the outside, which contrasts with previous models in several respects. This article reviews the behavior of site-directed mutants of Rhodobacter sphaeroides cytochrome c oxidase in the context of these different models. Studies of the internally located lysine 362 on the K channel and aspartate 132 on the D channel, indicate that D132, but not K362, is connected to the exterior region. Analysis of the externally located arginine pair, 481 and 482, and the Mg/Mn ligands, histidine 411 and aspartate 412, which are part of the hydrogen-bonded network that includes the heme propionates, indicates that alterations in this region do not strongly compromise proton pumping, but do influence the pH dependence of overall activity and the control of activity by the pH gradient. The results are suggestive of a region of 'sequestered' protons: beyond a major energetic gate, but selectively responsive to the external environment.

Mutations in the putative H-channel in the cytochrome c oxidase from Rhodobacter sphaeroides show that this channel is not important for proton conduction but reveal modulation of the properties of heme a.

As the final electron acceptor in the respiratory chain of eukaryotic and many prokaryotic organisms, cytochrome c oxidase catalyzes the reduction of oxygen to water, concomitantly generating a proton gradient. X-ray structures of two cytochrome c oxidases have been reported, and in each structure three possible pathways for proton translocation are indicated: the D-, K-, and H-channels. The putative H-channel is most clearly delineated in the bovine heart oxidase and has been proposed to be functionally important for the translocation of pumped protons in the mammalian oxidase [Yoshikawa et al. (1998) Science 280, 1723-1729]. In the present work, the functional importance of residues lining the putative H-channel in the oxidase from Rhodobacter sphaeroides are examined by site-directed mutagenesis. Mutants were generated in eight different sites and the enzymes have been purified and characterized. The results suggest that the H-channel is not functionally important in the prokaryotic oxidase, in agreement with the conclusion from previous work with the oxidase from Paracoccus denitrificans [Pfitzner et al. (1998) J. Biomembr. Bioenerg. 30, 89-93]. Each of the mutants in R. sphaeroides, with an exception at only one position, is enzymatically active and pumps protons in reconstituted proteoliposomes. This includes H456A, where in the P. denitrificans oxidase a leucine residue substituted for the corresponding residue resulted in inactive enzyme. The only mutations that result in completely inactive enzyme in the set examined in the R. sphaeroides oxidase are in R52, a residue that, along with Q471, appears to be hydrogen-bonded to the formyl group of heme a in the X-ray structures. To characterize the interactions between this residue and the heme group, resonance Raman spectra of the R52 mutants were obtained. The frequency of the heme a formyl stretching mode in the R52A mutant is characteristic of that seen in non-hydrogen-bonded model heme a complexes. Thus the data confirm the presence of hydrogen bonding between the heme a formyl group and the R52 side chain, as suggested from crystallographic data. In the R52K mutant, this hydrogen bonding is maintained by the lysine residue, and this mutant enzyme retains near wild-type activity. The heme a formyl frequency is also affected by mutation of Q471, confirming the X-ray models that show this residue also has hydrogen-bonding interactions with the formyl group. Unlike R52, however, Q471 does not appear to be critical for the enzyme function.

Definition of the interaction domain for cytochrome c on cytochrome c oxidase. I. Biochemical, spectral, and kinetic characterization of surface mutants in subunit ii of Rhodobacter sphaeroides cytochrome aa(3).

To determine the interaction site for cytochrome c (Cc) on cytochrome c oxidase (CcO), a number of conserved carboxyl residues in subunit II of Rhodobacter sphaeroides CcO were mutated to neutral forms. A highly conserved tryptophan, Trp(143), was also mutated to phenylalanine and alanine. Spectroscopic and metal analyses of the surface carboxyl mutants revealed no overall structural changes. The double mutants D188Q/E189N and D151Q/E152N exhibit similar steady-state kinetic behavior as wild-type oxidase with horse Cc and R. sphaeroides Cc(2), showing that these residues are not involved in Cc binding. The single mutants E148Q, E157Q, D195N, and D214N have decreased activities and increased K(m) values, indicating they contribute to the Cc:CcO interface. However, their reactions with horse and R. sphaeroides Cc are different, as expected from the different distribution of surface lysines on these cytochromes c. Mutations at Trp(143) severely inhibit activity without changing the K(m) for Cc or disturbing the adjacent Cu(A) center. From these data, we identify a Cc binding area on CcO with Trp(143) and Asp(214) close to the site of electron transfer and Glu(148), Glu(157), and Asp(195) providing electrostatic guidance. The results are completely consistent with time-resolved kinetic measurements (Wang, K., Zhen, Y., Sadoski, R., Grinnell, S., Geren, L., Ferguson-Miller, S., Durham, B., and Millett, F. (1999) J. Biol. Chem. 274, 38042-38050) and computational docking analysis (Roberts, V. A., and Pique, M. E. (1999) J. Biol. Chem. 274, 38051-38060).

Definition of the interaction domain for cytochrome c on cytochrome c oxidase. Ii. Rapid kinetic analysis of electron transfer from cytochrome c to Rhodobacter sphaeroides cytochrome oxidase surface mutants.

The reaction between cytochrome c (Cc) and Rhodobacter sphaeroides cytochrome c oxidase (CcO) was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 55 (Ru-55-Cc). Flash photolysis of a 1:1 complex between Ru-55-Cc and CcO at low ionic strength results in electron transfer from photoreduced heme c to Cu(A) with an intracomplex rate constant of k(a) = 4 x 10(4) s(-1), followed by electron transfer from Cu(A) to heme a with a rate constant of k(b) = 9 x 10(4) s(-1). The effects of CcO surface mutations on the kinetics follow the order D214N > E157Q > E148Q > D195N > D151N/E152Q approximately D188N/E189Q approximately wild type, indicating that the acidic residues Asp(214), Glu(157), Glu(148), and Asp(195) on subunit II interact electrostatically with the lysines surrounding the heme crevice of Cc. Mutating the highly conserved tryptophan residue, Trp(143), to Phe or Ala decreased the intracomplex electron transfer rate constant k(a) by 450- and 1200-fold, respectively, without affecting the dissociation constant K(D). It therefore appears that the indole ring of Trp(143) mediates electron transfer from the heme group of Cc to Cu(A). These results are consistent with steady-state kinetic results (Zhen, Y., Hoganson, C. W., Babcock, G. T., and Ferguson-Miller, S. (1999) J. Biol. Chem. 274, 38032-38041) and a computational docking analysis (Roberts, V. A., and Pique, M. E. (1999) J. Biol. Chem. 274, 38051-38060).

Proton uptake controls electron transfer in cytochrome c oxidase.

In cytochrome c oxidase, a requirement for proton pumping is a tight coupling between electron and proton transfer, which could be accomplished if internal electron-transfer rates were controlled by uptake of protons. During reaction of the fully reduced enzyme with oxygen, concomitant with the "peroxy" to "oxoferryl" transition, internal transfer of the fourth electron from CuA to heme a has the same rate as proton uptake from the bulk solution (8,000 s-1). The question was therefore raised whether the proton uptake controls electron transfer or vice versa. To resolve this question, we have studied a site-specific mutant of the Rhodobacter sphaeroides enzyme in which methionine 263 (SU II), a CuA ligand, was replaced by leucine, which resulted in an increased redox potential of CuA. During reaction of the reduced mutant enzyme with O2, a proton was taken up at the same rate as in the wild-type enzyme (8,000 s-1), whereas electron transfer from CuA to heme a was impaired. Together with results from studies of the EQ(I-286) mutant enzyme, in which both proton uptake and electron transfer from CuA to heme a were blocked, the results from this study show that the CuA --> heme a electron transfer is controlled by the proton uptake and not vice versa. This mechanism prevents further electron transfer to heme a3-CuB before a proton is taken up, which assures a tight coupling of electron transfer to proton pumping.

Cytochrome c oxidase from eucaryotes but not from procaryotes is allosterically inhibited by ATP.

The activity of reconstituted cytochrome c oxidase from bovine heart but not from Rhodobacter sphaeroides is allosterically inhibited by intraliposomal ATP, which binds to subunit IV. The activity of cytochrome c oxidase of wild-type yeast and of a subunit VIa-deleted yeast mutant, measured with Tween 20-solubilized mitochondria in the presence of an ATP-regenerating system, was also allosterically inhibited by ATP, indicating the general validity of this mechanism of "respiratory control" in eucaryotic cytochrome c oxidases (Arnold and Kadenbach, Eur. J. Biochem. (1997) 249, 350-354). Deletion of subunit VIa changes the biphysic into monophysic kinetics of the yeast enzyme in the presence of ADP. A tenfold higher amount of horse heart cytochrome c, as compared to yeast cytochrome c, was required to relieve the ATP inhibition of the yeast enzyme.

Overexpression and purification of cytochrome c oxidase from Rhodobacter sphaeroides.

The aa3-type cytochrome c oxidase of Rhodobacter sphaeroides has been overexpressed up to seven fold over that in wild-type strains by engineering a multicopy plasmid with all the required oxidase genes and by establishing optimum growth conditions. The two operons containing the three structural genes and two assembly genes for cytochrome c oxidase were ligated into a pUC19 vector and reintroduced into several oxidase-deleted R. sphaeroides strains. Under conditions of relatively high pH and maximal aeration, high levels of expression were observed. A smaller expression vector, pBBR1MCS, and a fructose promoter (fruP)5 were found not to enhance cytochrome c oxidase expression in R. sphaeroides. An improved cytochrome c oxidase purification protocol is reported, which combines histidine elution from a nickel affinity column and anion-exchange chromatography, and results in a higher yield and purity than previously obtained.

Proton uptake and release in cytochrome c oxidase: separate pathways in time and space?

Analysis of mutant forms of cytochrome c oxidase in conjunction with knowledge from high resolution crystal structures is providing important clues as to the location and specificity of proton channels and the timing of proton movements with respect to electron transfer events. Mutant forms of Rhodobacter sphaeroides cytochrome c oxidase at the highly conserved aspartate 132, in the 'D-channel' and at lysine 362, in the 'K-channel', are compared with respect to the nature of their residual activity and their reactions with H2O2. The results argue for physical separation and specificity in these two proton input routes, due to their distinctive kinetics with peroxide and the apparent connection of the D-channel, but not the K-channel, to the proton exit pathway. The reversible nature and possible location of the exit pathway are discussed in the context of direct and indirect mechanisms of energy coupling.

Aspartate-407 in Rhodobacter sphaeroides cytochrome c oxidase is not required for proton pumping or manganese binding.

Several pathways for proton transport in cytochrome c oxidase have been proposed on the basis of mutational analysis and X-ray structure: at least one for moving "pumped" protons from the interior to exterior of the membrane and a separate route for transporting "substrate" protons from the interior to the binuclear metal center to combine with oxygen to make H2O. According to the crystal structures of cytochrome c oxidase, Asp407 (Rhodobacter sphaeroides numbering) is at the interface of subunit I and subunit II of the oxidase, in a negative patch proposed to be the proton exit site in a pumping pathway, as well as a possible ligand to Mg [Iwata et al. (1995) Nature 376, 660-669]. Three mutants at the Asp407 position of R. sphaeroides cytochrome oxidase, Asp407Ala, Asp407Asn, and Asp407Cys, have been purified and characterized. All showed electron transfer activity, and pH dependence of activity, similar to that of the wild type enzyme and no major structural changes, as evidenced by visible, EPR, and resonance Raman spectroscopy. When reconstituted into artificial vesicles, the purified mutants pumped protons with normal efficiency and responded to the membrane pH and electrical gradients in a manner similar to that of wild type. Furthermore, the EPR spectra and Mn quantitation analysis of mutants grown in high Mn indicated no significant alteration in the Mn/Mg site. These results suggest that Asp407 does not play a critical role in proton translocation or in Mn/Mg binding.

Site-directed mutagenesis of residues lining a putative proton transfer pathway in cytochrome c oxidase from Rhodobacter sphaeroides.

Several putative proton transfer pathways have been identified in the recent crystal structures of the cytochrome oxidases from Paracoccus denitrificans [Iwata et al. (1995) Nature 376, 660-669] and bovine [Tsukihara (1996) Science 272, 1138-1144]. A series of residues along one face of the amphiphilic transmembrane helix IV lie in one of these proton transfer pathways. The possible role of these residues in proton transfer was examined by site-directed mutagenesis. The three conserved residues of helix IV that have been implicated in the putative proton transfer pathway (Ser-201, Asn-207, and Thr-211) were individually changed to alanine. The mutants were purified, analyzed for steady-state turnover rate and proton pumping efficiency, and structurally probed with resonance Raman spectroscopy and FTIR difference spectroscopy. The mutation of Ser-201 to alanine decreased the enzyme turnover rate by half, and was therefore further characterized using EPR spectroscopy and rapid kinetic methods. The results demonstrate that none of these hydrophilic residues are essential for proton pumping or oxygen reduction activities, and suggest a model of redundant or flexible proton transfer pathways. Whereas previously reported mutants at the start of this putative channel (e.g., Asp-132-Asn) dramatically influence both enzyme turnover and coupling to proton pumping, the current work shows that this is not the case for all residues observed in this channel.

Fatty acids stimulate activity and restore respiratory control in a proton channel mutant of cytochrome c oxidase.

(1) Removal of a carboxyl at residue 132 of subunit I of Rhodobacter sphaeroides cytochrome c oxidase significantly inhibits electron transfer and makes proton pumping undetectable [Fetter et al. (1995) Proc. Natl. Acad. Sci. USA 92, 1604-1608]. When reconstituted into phospholipid vesicles (COV), wild-type oxidase shows respiratory control that is partially released by either valimomycin or nigericin and fully released by the two ionophores combined. Under the same conditions, the D132A mutant COV show anomalous ionophore responses, including inhibition by valinomycin or by CCCP. Nevertheless, oxidase activity results in development of a similar membrane potential in COV containing either wild-type or D132A oxidase, and the ionophore responses of the membrane potential are similar for both enzymes. (2) Long chain fatty acids such as arachidonic acid, but not fatty alcohols, stimulate steady-state electron transfer activity 3-7-fold, with either detergent-solubilized (purified) D132A oxidase or the reconstituted form. The effect is specific for this mutant and is not seen with wild-type or other mutants of similar overall activity. Arachidonate-treated D132A COV show normal ionophore responses to valinomycin and nigericin and full release of respiration in presence of both ionophores or of CCCP. Thus, arachidonate and some other fatty acids abolish the ionophore anomalies seen when the D132A enzyme is reconstituted in their absence. (3) Fatty acid addition does not restore proton pumping, likely because fatty acids also induce proton permeability and some degree of uncoupling. A model of D132A function is presented and possible roles for the fatty acids in 'chemical rescue' of the mutant are discussed.

Polar residues in helix VIII of subunit I of cytochrome c oxidase influence the activity and the structure of the active site.

The aa3-type cytochrome c oxidase from Rhodobacter sphaeroides is closely related to eukaryotic cytochrome c oxidases. Analysis of site-directed mutants identified the ligands of heme a, heme a3, and CuB [Hosler et al. (1993) J. Bioenerg. Biomembr. 25, 121-133], which have been confirmed by high-resolution structures of homologous oxidases [Iwata et al. (1995) Nature 376, 660; Tsukihara et al. (1995) Science 269, 1069; (1996) 272, 1136]. Since the protons used to form water originate from the inner side of the membrane, and the heme a3-CuB center is located near the outer surface, the protein must convey these substrate protons to the oxygen reduction site. Transmembrane helix VIII in subunit I is close to this site and contains several conserved polar residues that could function in a rate-determining proton relay system. To test this role, apolar residues were substituted for T352, T359, and K362 in helix VIII and the mutants were characterized in terms of activity and structure. Mutation of T352, near CuB, strongly decreases enzyme activity and disrupts the spectral properties of the heme a3-CuB center. Mutation of T359, below heme a3, substantially reduces oxidase activity with only minor effects on metal center structure. Two mutations of K362, approximately 15 A below the axial ligand of heme a3, are inactive, make heme a3 difficult to reduce, and cause changes in the resonance Raman signal specific for the iron-histidine bond to heme a3. The results are consistent with a key role for T352, T359, and K362 in oxidase activity and with the involvement of T359 and K362 in proton transfer through a relay system now plausibly identified in the crystal structure. However, the characteristics of the K362 mutants raise some questions about the assignment of this as the substrate proton channel.

Protein structure: proton-pumping oxidases.

The crystal structures of two cytochrome c oxidases, one bacterial and one mammalian, offer insights into their roles in oxygen chemistry and as proton pumps.