Báo cáo khoa học: Defining the QP-site of Escherichia coli fumarate reductase by site-directed mutagenesis, fluorescence quench titrations and EPR spectroscopy

Defining the Q_p-site of Escherichia coli fumarate reductase by site-directed mutagenesis, fluorescence quench titrations and EPR spectroscopy

Tác giả: Richard A. Rothery, Andrea M. Seime, A.-M. Caroline Spiers, Elena Maklashina, Imke Schröder, Robert P. Gunsalus, Gary Cecchini and Joel H. Weiner

Lĩnh vực: Biochemistry, Biophysics, Microbiology, Immunology, Molecular Genetics

Nội dung tài liệu: Nghiên cứu này tập trung vào việc xác định vị trí liên kết quinone (Q_p-site) của enzyme fumarate reductase (FrdABCD) trong màng tế bào của vi khuẩn Escherichia coli. Thông qua các phương pháp đột biến định hướng, chuẩn độ bằng kỹ thuật huỳnh quang quench (FQ) và phổ cộng hưởng điện tử (EPR), các nhà nghiên cứu đã khảo sát ảnh hưởng của các đột biến tại các vị trí khác nhau lên khả năng liên kết của quinone và hoạt tính của enzyme. Kết quả cho thấy Q_p-site được định hình bởi các gốc axit amin từ các tiểu đơn vị FrdB, FrdC và FrdD, và có mối tương quan chặt chẽ với cụm [3Fe-4S] của FrdB. Nghiên cứu cũng nhấn mạnh sự tồn tại của một Q-site duy nhất, có khả năng hoạt động và phân ly, đóng vai trò trung tâm trong quá trình oxy hóa menaquinol.

Mục lục chi tiết:

• Introduction
• Keywords
• Correspondence
• Abbreviations
• Defining the Qp-site of Escherichia coli fumarate reductase by site-directed mutagenesis, fluorescence quench titrations and EPR spectroscopy
• We have used fluorescence quench titrations, EPR spectroscopy and steady-state kinetics to study the effects of site-directed mutants of FrdB, FrdC and FrdD on the proximal menaquinol (MQH2) binding site (QP) of Escherichia coli fumarate reductase (FrdABCD) in cytoplasmic membrane preparations.
• Fluorescence quench (FQ) titrations with the fluorophore and MQH2 analog 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) indicate that the Qp site is defined by residues from FrdB, FrdC and FrdD.
• In FQ titrations, wild-type FrdABCD binds HOQNO with an apparent Ka of 2.5 nm, and the following mutations significantly increase this value: FrdB-T205H (Ka = 39 nm); FrdB-V207C (Ka = 20 nm); FrdC-E29L (Ka = 25 nm); FrdC-W86R (no detectable binding); and FrdD-H80K (Ka = 20 nm).
• In all titrations performed, data were fitted to a monophasic binding equation, indicating that no additional high-affinity HOQNO binding sites exist in FrdABCD.
• In all cases where HOQNO binding is detectable by FQ titration, it can also be observed by EPR spectroscopy.
• Steady-state kinetic studies of fumarate-dependent quinol oxidation indicate that there is a correlation between effects on HOQNO binding and effects on the observed Km and keat values, except in the FrdC-E29L mutant, in which HOQNO binding is observed, but no enzyme turnover is detected.
• In this case, EPR studies indicate that the lack of activity arises because the enzyme can only remove one electron from reduced MQH2, resulting in it being trapped in a form with a bound menasemiquinone radical anion.
• Overall, the data support a model for FrdABCD in which there is a single redox-active and dissociable Q-site.
• Escherichia coli, when grown anaerobically with fumarate as the respiratory oxidant, develops a respiratory chain terminated by a membrane-bound menaquinol:fumarate oxidoreductase (FrdABCD) [1,2].
• The enzyme comprises a catalytic dimer of the FrdA (65.8 kDa) and FrdB (27 kDa) subunits that is anchored to the inner surface of the cytoplasmic membrane by two small hydrophobic membrane-anchor
• subunits, FrdC (15 kDa) and FrdD (13.1 kDa).
• The crystal structure of FrdABCD has been reported at 3.3 Å resolution [3,4], and has an overall architecture similar to that of the E. coli complex II homolog SdhCDAB (succinate:ubiquinone oxidoreductase) [5,6].
• Each enzyme contains a single FAD that is covalently bound to the catalytic subunit (FrdA/SdhA) and three [Fe-S] clusters (a [2Fe-2S] cluster, a [4Fe-4S] cluster, and a [3Fe-4S] cluster) coordinated by the electron-transfer subunit (FrdB/SdhB) [1].
• However, important differences exist between the membrane-intrinsic domains of the two enzymes [1,7].
• The membrane-intrinsic domain of SdhCDAB coordinates a single heme b (b556) that is sandwiched between the SdhC and SdhD subunits [8,9].
• Quinone binding and reduction is believed to take place in the region between the heme and the [3Fe-4S] cluster of SdhB [1,6].
• In the case of FrdABCD, the membrane-intrinsic domain does not contain heme, but instead contains two menaquinones at discreet sites in the crystallized form of the enzyme [3,4].
• In both enzymes, despite the available structures, the number of functional quinone/quinol binding sites has yet to be unequivocally determined.
• The menaquinones identified in the crystal structure of FrdABCD [3] are located at sites towards the inner (cytoplasmic) and outer (periplasmic) sides of the membrane-intrinsic domain of the enzyme (FrdCD).
• One site, the Qp site (the proximal Q-site), is located in the interface region between the FrdCD subunits and the [3Fe-4S] cluster coordinating region of FrdB on the cytoplasmic side of the membrane.
• The other site, the QD site (the distal Q-site) is located approximately 25 Å from the Qp site on the opposite (periplasmic) side of the membrane [3,10].
• The relatively large distance between the two sites may preclude direct electron-transfer through the protein medium, which is believed to be limited to a distance of approximately 14 Å [11].
• However, a third region of electron density has been identified recently between the Qp and QD sites (the ‘M’ site), and is centered approximately 13 Å from each Q-site [4].
• If this electron density corresponds to an additional electron-transferring cofactor, it could provide a conduit for electron-transfer from the QD site to the Qp site.
• However, analyses of the bioenergetics of respiratory growth of E. coli on fumarate indicate that FrdABCD turnover does not produce a transmembrane electrochemical potential [12], suggesting the presence of a single dissociable and redox-active Q-site that is formally located on the cytoplasmic side of the membrane.
• Menaquinol (MQH2) oxidation by FrdABCD has been studied using a combination of site-directed mutagenesis, enzymology, EPR spectroscopy and X-ray crystallography.
• Initial mutagenesis studies suggested that there may be two Q-sites present – a polar Qe site (equivalent to the Qp site), and an apolar QA site (equivalent to the QD site) [13-15].
• Investigation of the steady-state kinetics of quinol-dependent fumarate reduction by FrdABCD suggests that MQH2 binding and oxidation occur at a single site [16].
• Kinetic studies carried out in the presence of HOQNO or alkylated dinitrophenol derivatives also support the presence of a single MQH2 oxidation site [17].
• By exploiting the fluorescent properties of HOQNO in fluorescence quench (FQ) titrations, we determined that this inhibitor binds at a single high-affinity site within FrdABCD [18,19].
• EPR studies indicate that this high-affinity site is conformationally linked to the [3Fe-4S] cluster of FrdB [18].
• The emerging hypothesis that there is a single site for MQH2 or HOQNO binding has been complicated recently by the observation in crystallographic studies that the QD site is unoccupied when HOQNO or a dinitrophenol derivative is bound at the Qp site [4].
• Given the available structural information on FrdABCD, it would therefore be of interest to examine the effects of a range of site-directed mutants on the HOQNO binding properties and enzymology of the enzyme.
• In this paper, we evaluate the effects of mutation of amino acid residues located in the vicinity of the Qp site on HOQNO binding to FrdABCD.
• We have determined the effect of each mutation on HOQNO binding detected by FQ titration and EPR spectroscopy.
• We have also investigated the effects the mutants have on the steady-state kinetics of fumarate-dependent quinol oxidation.
• Results
• Selection of mutants of FrdB, FrdC and FrdD
• The following residues are located within approximately 5 Å of the menaquinone (MQ) observed at the Qp-site in the structure of FrdABCD: T205, F206, Q225 and K228 from FrdB; R28, E29, W86, L89 and A93 from FrdC; and W14, F17, G18, H80, R81 and H84 from FrdD [3,4,10].
• Site-directed mutants of some of these residues have been generated and partially characterized, including the following: FrdC-E29L [14,20], FrdC-W86R, FrdD-H80K and FrdD-H84K [14].
• In the context of this study, mutants of the following residues located at a slightly greater distance from the Qp site are also potentially of interest: FrdB-V207 (≈ 8 Å from Qp, a FrdB-V207C mutant) [21], and FrdC-A32 (≈ 9 Å from Qp, a FrdC-A32V mutant) [14].
• At an even greater distance away from the Qp site is FrdC-F38 (≈ 18 Å), a mutation at this position (FrdC-F38M [14]), would be expected to have little effect on MQH2 binding and
• oxidation.
• Finally, we generated a mutant of FrdB-T205 (FrdB-T205H) to assess the role of the [3Fe-4S] cluster binding domain of FrdB in defining the Qp-site.
• This residue is sandwiched between the [3Fe-4S] cluster and the Qp site.
• All eight mutant enzymes were studied to assess the effects of the mutations on MQH2 binding using FQ titrations, EPR spectroscopy and steady-state kinetic studies.
• The locations of all the mutated residues located within ≈10 Å of the Qp site are illustrated in Fig. 1.
• HOQNO has a very similar structure to that of MQ, and as a result appears to bind to the Qp site in an almost identical way (compare Fig. 1A and B with C and D).
• This similarity in both structure and binding renders HOQNO an excellent inhibitor with which to characterize the Qp site of FrdABCD.
• FQ titrations of HOQNO binding to mutant FrdABCD
• HOQNO is a close structural analog of MQH2/MQ and is a very potent inhibitor of FrdABCD [16,18].
• When excited at 341 nm, free HOQNO in aqueous solution fluoresces with an emission wavelength of 479 nm.
• Its fluorescence is completely quenched when bound to FrdABCD and certain other E. coli respiratory chain enzymes (including dimethylsulfoxide reductase and nitrate reductase A [18,19,22-24]).
• This enables its binding to a Q-site to be analyzed by FQ titration.
• Figure 2 shows representative titrations of membranes containing the wild-type and mutant enzymes studied herein.
• Data for all of the mutants is presented in Table 1.
• DW35 membranes lacking FrdABCD (Fig. 2A) do not exhibit high-affinity HOQNO binding.
• The following FrdABCD mutants bind HOQNO with Ka values equivalent to that of the wild-type enzyme (Ka = 2.5 nm; Fig. 2B): FrdC-A32V (2.5 nm; not shown), FrdC-F38M (2.5 nm; not shown) and FrdD-H84K (3.0 nm, not shown).
• At the opposite extreme, it is clear that the FrdC-W86R mutant does not exhibit high-affinity HOQNO binding (Fig. 2E).
• This mutant appears to have a similar phenotype to that of the previously reported FrdC-H82R mutant [18,25].
• Intermediate effects are observed with the following mutants: FrdB-T205H (Ka = 39 nm; Fig. 2C), FrdB-V207C (20 nm; not shown), FrdC-E29L (25 nm; Fig. 2D) and FrdD-H80K (20 nm; Fig. 2F).
• Based on these observations and the FrdABCD structure [3,4], it is clear that residues from FrdB, FrdC and FrdD play important roles in defining the Qp site.
• In every case where binding is detected, the data can be fitted to an equation (Eqn 1) describing noncooperative binding at a single site within FrdABCD.
• Table 1 shows the calculated specific concentration of HOQNO binding sites for each mutant in which binding is detected by FQ titration.
• It also shows the concentration of FrdABCD calculated by EPR spin quantitation of both the [2Fe-2S] and [3Fe-4S] clusters.
• In each case, the estimated number of Q-sites per enzyme is very close to unity, indicating that HOQNO binding occurs at a single site within FrdABCD.
• Based on enzymes that bind HOQNO, 1.02 ± 0.12 sites were observed per [3Fe-4S] cluster and 1.05 ± 0.09 sites were observed per [2Fe-2S] cluster.
• Detection of HOQNO binding by EPR spectroscopy
• Figure 3 shows the effect of HOQNO on the EPR spectrum around g = 2.0 of ferricyanide-oxidized HB101 membrane samples containing wild-type and mutant FrdABCD.
• EPR spectra of membranes lacking overexpressed FrdABCD exhibit low-intensity features around g = 2.0 upon which HOQNO has little effect (Fig. 3A).
• Spectra of membranes containing overexpressed wild-type FrdABCD exhibit the EPR spectrum of its oxidized [3Fe-4S] cluster (Fig. 3B).
• This spectrum is nearly isotropic with a peak at g = 2.02 (gz) and a broad trough immediately up-field.
• As has been reported previously [18,20], addition of HOQNO elicits the observation of an additional peak-trough at approximately g = 1.98 (gxy).
• Both of the FrdB mutants studied herein (FrdB-T205H and FrdB-V207C) have significant effects on the EPR properties of FrdABCD.
• In the case of the FrdB-T205H mutant, the [3Fe-4S] cluster line-shape is narrower than that of the wild-type (note the position of the trough in the spectrum without HOQNO; Fig. 3C).
• As is the case for the wild-type enzyme, addition of HOQNO results in the resolution of a peak-trough on the high-field side of the g = 2.02 peak.
• This peak-trough is centered at a g-value reflecting the narrower spectrum of the [3Fe-4S] cluster in the
• FrdB-T205H mutant in the absence of inhibitor (gxy = 2.0 in the presence of inhibitor rather than at 1.98).
• Figure 3D shows the spectrum of oxidized mem- branes containing overexpressed FrdB-V207C mutant enzyme.
• In agreement with Manadori et al. [21], little or no [3Fe-4S] cluster is assembled into this mutant enzyme (compare Fig. 3A and D), and therefore HOQNO binding cannot be detected by its perturbation of the EPR spectrum of the oxidized enzyme (see below).
• In contrast to the results of Hägerhäll et al. [20], the EPR experiments reported herein indicate that HO- QNO elicits an effect on the EPR line-shape of the [3Fe-4S] cluster of the FrdC-E29L mutant enzyme (Fig. 3E).
• This result is consistent with the observation of HO