Models for structure and function in quinone-binding sites: the

Quinone-Binding Sites in Membrane Proteins

Models for structure and function in quinone-binding sites: the Escherichia coli quinol oxidase, cytochrome bo, L. Murray, R. H. Pires, S. F. Hastings and W. J. lngledew School of Biological and Medical Sciences, University of St. Andrews, St. Andrews, Scotland, KY I 6 9AL, U.K. cytochrome uu3, in all aspects of its structure and function, except that it lacks the cytochrome c binding and the Cu, prosthetic group and instead has quinone-binding site(s) [S]. T h e putative oxidation of quinol by ferrihaem (cytochrome b ) in sequential one-electron steps would require the stabilization of semiquinone. However, mechanisms involving two quinol-binding sites have also been proposed [6-81. Experimentally, the presence of a locum that highly stabilizes ubisemiquinone with respect to free ubisemiquinone is well established in the complex [9]. If there is a second site, for which there is evidence, it is not one at which the semiquinone radical is thermodynamically stabilized. Structural information is presented below on a putative quinol-binding site in the bo, oxidase.

Introduction Quinone-binding sites are obligatory in quinoneutilizing respiratory and photosynthetic complexes. I n addition to their importance in catalysis in electron-transfer systems involved in energy transduction, these loci are the sites of action of numerous inhibitors, including pesticides, herbicides and antibiotics [l]. T h e binding sites are of three types ; acceptor, donor and pair-splitting loci. In the first two types of site the potentials of the two half reactions have to be approximately equal, as the electrons are fed to/from the same electron donor/acceptor and this requires stabilization of the semiquinone. Since the stability constant of free ubisemiquinone at neutral p H is x lo-'', the binding of the semiquinone must be at least x lo5 times as tight as the other two species to achieve a stability constant of unity [2,3]. Tight binding of the semiquinone also allows the site to confine the reactive semiquinone species, conferring kinetic and thermodynamic stabilization. Pair-splitting loci, such as that found in the quinol cytochrome c reductase complex, do not stabilize the semiquinone, as this is unnecessary for the thermodynamics of the reaction [4]. T h e pair-splitting site for quinol oxidation (Q, or Q,) by quinol cytochrome c reductase facilitates the oxidation of quinol to quinone by two different electron carriers, with widely separated redox potentials, functioning as acceptors [4]. T h e spatial separation of the acceptors and their gross thermodynamic difference requires a site with different properties from the sites of quinone reduction. T h e x 300-400 mV separation in the potentials of the acceptors effectively removes the requirement for thermodynamic stabilization of the semiquinone state at this site. T h e amino acid sequences of many enzymes with quinone-binding sites have been published. In addition, a small number of crystal structures are available. No general sequence motif has been recognized for quinone-binding sites, but a structural motif is beginning to emerge (see below). T h e Escherichiu coli quinol oxidase cytochrome bo, is closely related to the cytochrome c oxidase,

General models for the structure of quinone-binding sites Semiquinone-stabilizingsites T h e crystal structures of sites at which semiquinone stabilization occurs are available from six species of photosynthetic reaction centre (each of which contains both a Q, and a Q, quinonebinding site) and two quinol cytochrome c reductase complexes (Qi sites) [1&19]. Analysis of these sites reveals common structural elements. In each structure the quinone ring is seen to be stabilized by a hydrophobic pocket in the protein matrix. T h e dimensions of the pocket are x 7.5 A between the hydrophobic residues above and 9.5 A bebelow the quinone-ring plane and tween the groups hydrogen-bonding the quinone oxygens. T h e quinone-ring region interacts with residues of the pocket, which extends to enclose the first isoprenoid units of the tail which are bound through hydrophobic interactions (two units in Q, and one unit in Q,J. In the photosynthetic reaction Q, centres, conserved histidine, isoleucine and tryptophan residues bind the quinone ring, while in the quinol cytcohrome c reductase complex the histidine is retained but phenylalanine replaces the tryptophan and a side group of a haem replaces the isoleucine in the same spatial and chemical arrangement. T h e structures of the quinone-binding sites from the six resolved

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Q, sites also show the same structural pattern. Here, binding of the quinone ring also occurs in a pocket with interactions from conserved histidine, isoleucine and phenylalanine residues. However, in the Q, site the pocket extends to only enclose the first isoprenoid unit of the tail. The isoleucine and tryptophan residues provide hydrophobic interactions above and below the quinone ring, whereas the histidine residue forms a hydrogen bond with the quinone oxygen distal to the isoprenoid side chain. The fourth position of the tetrad is that hydrogen-bonding the quinone-ring oxygen (adjacent to the isoprenoid side chain). In the Q, sites, this bond is formed with the backbone nitrogen of an alanine residue, whereas in the Q, sites the bond is formed with the -OH side chain of a serine residue. These are summarized in Figure 1. The quinone-binding sites of Q, and Q, have differing roles. Q, accepts an electron from bacteriophytin and within l o o p the electron is transferred to Q,. This process is repeated, with Q, picking up a second electron and two protons. The ubiquinol dissociates from Q, and the site is refilled from the membrane quinone pool. Hence, both sites are capable of stabilizing the ubisemiquinone radical but in the Q, site the quinone/ quinol can readily exchange with the membrane quinone pool. The Q, sites of chicken and bovine quinol cytochrome c reductase have a similar structure to those of the Q, and Q, sites. Here a phenylalanine

residue and a haem side chain provide hydrophobic interactions above and below the plane of the ring. His-202 forms a hydrogen bond with the oxygen distal from the isoprenoid chain, while Ser-36 and Asp-229 hydrogen-bond to the remaining oxygen. Alignment of the binding residues in the Q, site with those from Q, and Q, show that the Q, site appears to be a mirror image of the Q, and Q, sites (Figure 1B). When aligned, the phenylalanine residue occupies the position of the isoleucine in Q, and Q,, while the haem side chain occupies the position of the tryptophan in Q, and phenylalanine in Q,. A summary of the quinonebinding residues of the Q,, Q, and Q, sites can be found in Table 1. Although quinone occupies the sites in the published crystal structures, some of these sites must have the capacity to bind the semiquinone anion much more tightly ( lo5-fold) than the quinone. This ability will distinguish the different types of binding site. It is normally the semiquinone anion which is bound. In each site there exists the possibility of multiple hydrogen-bond formation to the quinone oxygens. In the Q, sites an additional hydrogen bond may form with the residue Thr-222, while in the Q, site hydrogen bonds may form with the backbone nitrogens of Ile-224 and Gly-225. An electrostatic bond would be strongest in stabilizing the semiquinone anion but strong hydrogen-bonding may suffice and the strongest of these can be formed between a serine

Figure I Structures of the semiquinone-stabilization sites from the QA and Qe sites of the photosynthetic reaction centre of Rhodobacter sphaeroides and the chicken heart quinol cytochrome c reductase (A) Alignment of quinone-binding residues of R sphaeruides QA and Qe sites. Taken from the PDB file IAIG. Hydrophobic interactions above and below the quinone ring are provided by Trp(W)-252, lle(l)-263 (QA) and Phe(F)-216 and lle-229 (QJ with hydrogen bonding t o His(H)-2 I 9 and Ala(A)-260 (QA) and His- I90 and Ser(S)-223 (QJ. (B) Structurr of the chicken Qi Site. Taken from PDB file I BCC. Phe-22 I and the haem provide hydrophobic interaction above and below the quinone ring with His-202 and Ser-36/Asp(D)-229 hydrogenbonding t o the quinone oxygens.

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Table I

Summary of the quinone - binding residues, taken from available structures of photosyntheticQ, and QBsites, and quinol cytochrome c reductase Qi (b) denotes hydrogen - bonding t o a peptide - NH group. Possibilitiesfor additional H - bonding are also shown. Contact I and 2 are residues providing hydrophobic interactions above and below the plane of the quinone ring. Distal and proximal 0 refen to the quinone oxygens distal and proximal to the isoprenoid tail.

PDB code

Q site

Species

Contact I

Contact 2

Distal 0 H -bond

Proximal 0 H -bond

IAIG

Q A

W-252

1-263

H-2 I9

A-260(b)

Q B

Rhodobacter sphaeroides R. sphaeroides R. sphaeroides R. sphaeroides R. capsulatus R. vindis Chicken Bovine R. sphaeroides

W-252 W-252 W-252 W-252 "-250 F-22 I F-220 F-2 I 6

1-263 1-263 1-263 1-263 1-263 Haem2 Haem2 1-229

H-2 I9 H-2 I9 H-2 I 9 H-2 I7 H-2 I7 H-202 H-20 I H- I 90

PCR

Q0

R. sphaeroides

F-2 I 6

1-229

H- I90

YST

Q B

R. sphaeroides

F-2 I 6

1-229

H- I90

PSS

Q 0

R. sphaeroides

F-2 I 6

1-229

H- I90

CLT

Q 0

R. capsulatus

F-2 I 6

1-229

H- I90

I PRC

Q B

R. viridis

F-2 I6

1-229

H- I90

A-260(b) A-260(b) A-260(b) A-25 8 (b) A-25 8 (b) 5-36, D229 S-36, D229 5-223, 1-224(b), G-225(b) S-223, 1-224(b), G-225(b) S-223, 1-224(b), G-225(b) S-223, 1-224(b), G-225(b) 5-223, 1-224(b), G-225(b) 5-223, 1-224(b), G-225(b)

I PCR I YST PSS CLT PRC BCC BE3 AIG

Q A

Q A Q A Q A Q A

Q, Q,

-OH and 0- in linear alignment. Because of the pH-independence of the potential of most Q + Qtransitions, protonation of the histidine to allow an electrostatic bond is not likely.

speculative. It is rational that the site cannot bind the semiquinone radical anion as effectively as the equivalent residues in semiquinone-stabilizing sites (as the radical is not tightly bound at this site) and in theory this could be the only difference between the two types of site. Comparison of this site before and after binding of stigmatellin reveals that the site expands as the stigmatellin binds to the site, indicating the site's substantial flexibility. However, comparisons of the Q, site of Rhodobacter sphaeroides with and without the bound ubiquinone [18] show no apparent expansion of the Q, site on binding of the ubiquinone molecule.

Pair-splitting sites No structures are yet available showing a quinone bound in a pair-splitting site, but a structure of a chicken quinol cytochrome c reductase complex with the Q,-site inhibitor stigmatellin bound is available [ 191. The stigmatellin-binding site is thought to overlap that of the quinone-binding site and a consideration of the structure indicates this; comparisons of the structure of the pocket in which stigmatellin binds with the structures of other Q-sites are informative. A pocket is observed with similar potential hydrophobic interactions above and below the quinone ring but the pocket is open at both ends and assigning putative hydrogen-bonding of the two quinone oxygens is too

The quinone-binding sites of cytochrome bo, Modelling subunit I1 of the cytochrome bo, based on comparisons with the crystal structures of a bacterial cytochrome aa, [20] (PDB file l A R l ) , bovine cytochrome aa, [21] (PDB file 1OCC) and the crystal structure of a fragment of cytochrome

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with this model (S. E. J. Rigby, P. Heathcote, S. F. Hastings and W. J. Ingledew, unpublished work). T h e quinone-binding elements of photosynthetic Q, and Q, sites can be aligned with the proposed quinone-binding site in the cytochrome bo,. T h e Trp-136 residue occupies the same spatial position as the tryptophan and phenylalanine in the Q, and QB sites, Val-170 aligns with the isoleucines of Q, and QB, and the serine -OH side chain aligns with the histidine rings of Q, and Q,. This composite is shown in Figure 2. There have been suggestions that two quinone-binding sites are present in cytochrome bo, ; a low-affinity site which does not stabilize semiquinone (Q,) and so functions as a pair-splitting site donating one electron to cytochrome b and one to a high-affinity quinone-binding site (Q,) [6]. At p H 7.0 the redox potentials of the acceptors are 130 mV (cytochrome b) and 25 mV (Q -+ Q-) [4], not as widely separated in potential as the pairsplitting site in quinol cytochrome c reductase ( x 300 mV). At present there is insufficient structural data on the location of a second Q site to allow modelling of it. In addition, a portion of the subunit I I of the bo, cannot be modelled as there is no similar sequence in the aa, oxidases to act as a template. Cytochrome bo, does tightly bind quinone. T h e site we have modelled here is more like the Qr site of quinol cytochrome c reductase, in that the pocket is shallow and cannot accommodate more than part of the first isoprenoid unit. Therefore, it may not be a tight site like the Q, site, in which the pocket accommodates the first two units of the isoprenoid side chain.

bo, subunit I1 [22] (PDB file 1CYW) reveals a putative quinone-binding site. T h e putative quinone-binding pocket of cytochrome bo, has Trp136 and Val-170 providing hydrophobic interactions above and below the plane of the quinone ring. T h e oxygen distal to the isoprenoid tail could be stabilized by a hydrogen bond to the backbone nitrogen of Phe-215, with the remaining oxygen hydrogen-bonded to Ser-169 and possibly Asp135. T h e site exists at the interface between subunits I and I1 and is located directly above the haems of subunit I. Only the first isoprenoid unit of the tail is stabilized in the site. T h e remainder of the tail could be stabilized in a cleft between transmembrane helices of subunit I. T h e loose binding of the tail suggests that the quinone in this site can exchange with the membrane quinone pool. Electron-nuclear double resonance (ENDOR) and electron spin-echo envelope modulation (ESEEM) data on the stabilized ubisemiquinone of cytochrome bo, suggest that a peptide nitrogen and either a serine or threonine -OH group form hydrogen bonds with the stabilized ubisemiquinone radical, observations consistent

+

Figure 2 Alignment of the quinone-binding residues of the putative cytochrome bo, quinone-binding site with those of the QA and Qg sites of R sphaeroides Showing the similar spatial arrangement of the binding residues. QA and QB sites are taken from the PDB file I AIG. The fint residue label is for subunit II ofthe bo, oxidase. The second and third labels are for residues of the R. spoeroides QA and QB sites respectively.

+

EPR studies on a stabilized semiquinone of cytochrome bo, We have reported the properties of an ubisemiquinone radical in appropriately poised samples of purified enzyme reconstituted with excess ubiquinone [4]. T h e ubisemiquinone is highly stabilized with respect to free ubisemiquinone ; significant free radical can be observed, even at p H 7.0, whereas at p H 9.0 the stability constant is 5-10. T h e pH-dependence of the stability constant indicates that the anionic form of the semiquinone predominates above p H 7.5. T h e line width of the EPR spectrum is x 0.9 m T , which is consistent with a ubisemiquinone anion. In comparison with other respiratory chain Q-' species that have been described, the relaxation rate in the presence of

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reduced haems appears comparable with magnetically isolated Q-' radicals. Partially resolved splittings of z 0.4 m T can be observed in the spectrum of the semiquinone anion bound to cytochrome bo,. These studies have been extended to look at the orientation dependence of the EPR spectra in oriented multilayers. Spectra show, in oriented samples, a change in the position of the zero crossing of the signal [23] with angle to the magnetic field. This can be used to determine the orientation of the semiquinone ring plane due to the magnitude of theg-anisotropy, even at X-band frequency [24]. T h e spectra show marked orientation dependencies manifested as changes in line shape and g value (results not shown). T h e shape of the EPR signal is determined by the g-tensor and by hyperfine interactions, both of which are orientation-dependent. T h e hyperfine interactions are approximately symmetrical about the field position, thus the only parameter that determines the zero crossing is the g-anisotropy [25]. Preliminary analyses indicate that the quinone plane (g, corresponding to the in-plane axis perpendicular to the x-axis, which is in-plane through the quinone oxygens) is at z 20" to the membrane plane and the x-axis is at z 75" to the membrane plane, but further studies using higher frequencies and more sophisicated spectroscopies are required to interpret the data more accurately. T h e quinone we have modelled in the putative loose Q site in cytochrome bo, lies with its x- and y-axes x 24" from the membrane plane, the zaxis perpendicular at 66".

7 8 9 10 II

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Received 19 March 1999

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