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The fully oxidized form of the cytochrome bd quinol oxidase from E. coli does not participate in the catalytic cycle: Direct evidence from rapid kinetics studies
FEBS Letters 582 (2008) 3705–3709
The fully oxidized form of the cytochrome bd quinol oxidase from E. coli does not participate in the catalytic cycle: Direct evidence from rapid kinetics studies Ke Yanga, Vitaliy B. Borisovb, Alexander A. Konstantinovb, Robert B. Gennisa,* b
a Department of Biochemistry, University of Illinois, 600, Southgoodwin Avenue, Urbana, IL 61801, USA A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russia
Received 28 July 2008; revised 13 September 2008; accepted 16 September 2008 Available online 26 September 2008 Edited by Miguel De la Rosa
Abstract Cytochrome bd catalyzes the two-electron oxidation of either ubiquinol or menaquinol and the four-electron reduction of O2 to H2O. In the current work, the rates of reduction of the fully oxidized and oxoferryl forms of the enzyme by the 2-electron donor ubiquinol-1 and single electron donor N,N,N 0 ,N 0 -tetramethyl-p-phenylendiamine (TMPD) have been examined by stopped-flow techniques. Reduction of the all-ferric form of the enzyme is 1000-fold slower than required for a step in the catalytic cycle, whereas the observed rates of reduction of the oxoferryl and singly-reduced forms of the cytochrome are consistent with the catalytic turnover. The data support models of the catalytic cycle which do not include the fully oxidized form of the enzyme as an intermediate. 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Cytochrome bd; Ubiquinol; Respiration
1. Introduction The cytochrome bd quinol oxidase is one of the two terminal oxidases in the respiratory chain of E. coli, and catalyzes the 2electron oxidation of either menaquinol or ubiquinol and the four-electron reduction of oxygen to water [1–5]. This enzyme is a member of a large family of respiratory oxidases that are found in many prokaryotes, both bacteria and archaea. There is no eukaryotic homologue, and the presence of bd-type oxidases in a number of human pathogens makes this enzyme a possible drug target. Cytochrome bd is a transmembrane heterodimer, with one copy each of two proteins coded by the genes CydA (subunit I; 58 kDa) and CydB (subunit II; 43 kDa) [6]. There is no sequence homology between cytochrome bd and the large superfamily of the heme–copper respiratory oxidases [7], and the bd-type oxidases do not contain copper [6]. During turnover, cytochrome bd does not pump protons but it generates a proton motive force due to the fact that the protons used in the reaction to make H2O come from * Corresponding author. Fax: +1 217 244 3186. E-mail address: [email protected] (R.B. Gennis).
Abbreviations: TMPD, N,N,N 0 ,N 0 -tetramethyl-p-phenylendiamine; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; UQH21, ubiquinol-1
the bacterial cytoplasm, whereas the protons from the oxidation of QH2, which is oxidized near the opposite side of the membrane, are released to the periplasm [8,9]. Cytochrome bd contains three heme prosthetic groups to transfer electrons from quinol to oxygen: heme b558, heme b595 and heme d. The low spin heme b558 is believed to be the entry site of electrons from quinol and is located near the putative quinol binding site. Heme d is the site where oxygen binds and is reduced to water. The role of the high spin heme b595 is ˚ from the high still not clear, but heme b595 is located about 10 A spin heme d [10] and minimally its role is to provide an electron to facilitate the reduction of oxygen bound to heme d. The membrane potential appears to be generated essentially by proton uptake accompanying the electron transfer from heme b558 to the oxygenated heme b595/heme d center, which generates a product with a high affinity for protons [8]. The isolated cytochrome bd appears to have a single binding site for quinols. Unlike the heme–copper oxidases that utilize ubiquinol as a substrate, such as cytochrome bo3 from E. coli, there is no evidence for a tightly bound quinone which does not readily exchange with the quinone pool in the membrane. Hence, the isolated cytochrome bd has no bound quinone and the fully reduced form of the enzyme contains three electrons for redox chemistry, corresponding to the ferrous forms of each of the three hemes. Four different states of heme d have been characterized spectroscopically in cytochrome bd. These are (1) ferric (Fe3+); (2) ferrous (Fe2+); (3) ferrous–oxy complex (Fe2+–O2) and (4) oxoferryl (Fe4+‚O2). A putative peroxy complex (supposedly, Fe3+–OOH) has been observed recently as a transient intermediate during oxidation of the fully reduced enzyme by O2 [11], but its structure requires further confirmation. Each of the other four forms of heme d can be generated as stable or metastable forms that can be examined and characterized. These forms suggest a sequence of events at heme d as O2 is converted to water Fe2þ ! Fe2þ O2 þO2
Fe4þ @O2
!
þe þHþ
!
Fe3þ OAOH
þe þ2Hþ H2 O
!
þe þHþ H2 O
Fe3þ ! Fe2þ þe
ð1Þ
The analysis of enzyme turnover, however, must take into account the redox status of the other two heme prosthetic groups, heme b558 and heme b595, which can exist in either the Fe2+ or Fe3+ states, and also the fact the ubiquinol is a two-electron donor.
0014-5793/$34.00 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.09.038
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The purpose of the current work is to directly test the proposal based on the steady-state kinetics studies [12,13] that 3þ 3þ the fully oxidized (b3þ 558 b595 d ) form of cytochrome bd may not be part of the catalytic cycle (Fig. 1). The model shown in Fig. 1 implies that the rate of reduction of the all-ferric form of the enzyme should be slow. Contrary to this proposal, fast reduction of heme d in the all-ferric enzyme was observed in pulse radiolysis studies [14]. The generation of a strong one-electron reductant in this experiment resulted in very rapid reduction of heme b558, equilibration within 10 ls between heme b558 and heme b595, followed by electron transfer to heme d with the rate constants of about 3400 s1 and 590 s1, for the ferric and the oxoferryl forms, respectively. The one-electron transfer to heme d in the ferrous–oxy form (Fe2+–O2) was about 100-fold slower than observed with the ferric and oxoferryl forms. Taken at face value, these data suggest that the all-ferric form should be reconsidered as a possible intermediate during steady state analysis, and raises questions about the catalytic competence of the ferrous–oxy complex as an intermediate. An important difference between the steady-state turnover and pulse radiolysis experiments is that the former normally utilizes a two-electron donor whereas the latter utilizes a one-electron reductant. This was pointed out by Kobayashi et al. [14], in reference to the slow single-electron transfer to the ferrous–oxy complex. In the current work, a rapid mixing/diode array spectrophotometer was used to monitor the rate of reaction of ubiquinol1 (UQH2-1) and of ascorbate-reduced N,N,N 0 ,N 0 -tetramethylp-phenylendiamine (TMPD) with the all-ferric form of cytochrome bd. The kinetics of these reactions were compared with the reactions with the oxoferryl form of the enzyme generated by transient oxidation of the fully-reduced cytochrome bd by a stoichiometric amount of oxygen. The results reveal very slow (seconds) reduction of the all-ferric enzyme by ubiquinol-1, whereas reduction of the oxoferryl form of cytochrome bd is at least
K. Yang et al. / FEBS Letters 582 (2008) 3705–3709
1000-fold more rapid and compatible with the turnover number of the enzyme. With a single-electron donor TMPD, the oxoferryl form of cytochrome bd is also a much better electron acceptor than the all-ferric form, however, the difference is much less pronounced than in the case of two-electron reductant UQH2-1. The data show directly that the all-ferric form of the enzyme is not likely to be part of the normal catalytic cycle.
2. Materials and methods 2.1. Strains and plasmids E. coli strain GO105 (cyd AB::kan, cyo, recA), which lacks both the cytochrome bo3 and cytochrome bd quinol oxidases [15], was used as the host strain for overexpressing the wild type cytochrome bd from plasmid pTK1 [15] which was introduced into the strain. 2.2. Cell growth and enzyme purification Large scale cell growth was carried out in 24 2 L flasks shaking at 220 rpm and 37 C using an Innova 4330 incubator shaker (New Brunswick Scientific) [16]. Cytochrome bd was purified as described previously [17]. The pooled fractions were concentrated using an Amicon concentrator with a 50 kDa molecular weight cut-off filter and then dialyzed three times against 50 mM sodium phosphate buffer, pH 7.8, containing 5 mM ethylenediaminetetraacetic acid (EDTA), 0.05% N-lauroylsarcosine. 2.3. Heme analysis The heme b content of purified cytochrome bd was measured by the pyridine hemochromogen assay, using an extinction coefficient for the wavelength pair 556.5540 nm, De556.5–540 = 24 mM1 cm1 [18]. The heme d content and enzyme concentration were determined from the absolute spectrum of the fully reduced enzyme, using the extinction coefficient De628–670 = 25 mM1 cm1 [8]. 2.4. Ubiquinol-1 and TMPD oxidase activity assays The steady state oxidase activity assays were performed as described previously by Zhang et al. [17]. The purified enzyme had a turnover number of about 1100 s1 with 100 lM UQH2-1 + 3 mM dithiothreitol (DTT) and about 160 s1 with 10 mM ascorbate + 0.5 mM TMPD.
Fig. 1. Catalytic cycle of ubiquinol-oxidase activity of cytochrome bd. The scheme is based on the work of Ju¨nemann et al. [12] as modified by Matsumoto et al. [13].
K. Yang et al. / FEBS Letters 582 (2008) 3705–3709
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2.5. UV–visible spectroscopic measurements Static absorbtion spectra were obtained with a UV-2101PC spectrophotometer (Shimadzu). 2.6. Stopped-flow experiments The stopped-flow experiments were performed at 20 C using an Applied Photophysics stopped flow with a photodiode array detector. The flow system was flushed thoroughly with argon-saturated buffer (50 mM sodium phosphate, 5 mM EDTA and 0.05% N-lauroylsarcosine, pH 7.8) before the experiments. The whole system was kept anaerobic by flowing argon during the experiments. The mixing ratio of the two solutions was 1:1. After mixing, 1600 spectra were collected in the 300–1200 nm range over a period ranging from 2 s to 1 min. At least three runs were performed for each time scale. The concentrations reported are the initial concentrations before mixing, if not stated otherwise. 2.7. Generation of the oxoferryl form of the enzyme Five micromolar of enzyme was fully reduced by 200 lM ubiquinone-1 + 6 mM dithiothreitol or by 1 mM TMPD + 20 mM ascorbate in an argon-saturated buffer (50 mM sodium phosphate, 5 mM EDTA and 0.05% N-lauroylsarcosine, pH 7.8). The fully reduced enzyme 2þ 2þ (b2þ 558 b595 d ) was then rapidly mixed in the stopped-flow apparatus with an equal volume of a solution containing approximately 5 lM O2 in the same buffer. The reduced cytochrome bd was rapidly oxidized by O2 yielding essentially the oxoferryl form of the enzyme 3þ 4þ 2 (b3þ 558 b595 d @O ), which was subsequently reduced by the excess reductant present in the solution. The oxidation reaction is too fast to resolve, but the tail end of the reduction kinetics could be observed spectroscopically. Spectroscopic analysis of the product of the reaction of O2 with the reduced enzyme showed that it contained approximately 80% oxoferryl and 20% ferrous–oxy complex (not shown). 2.8. Experiments with the all-ferric form of cytochrome bd Five micromolar of the ‘‘as isolated’’ cytochrome bd, mainly present 3þ 2þ as ferrous–oxy complex b3þ 558 b595 d AO2 , was incubated for 20 min with 16 lM tetrachlorobenzoquinone, a lipophilic strong oxidant [19], in argon-saturated buffer (50 mM sodium phosphate, 5 mM EDTA and 0.05% N-lauroylsarcosine, pH 7.8). The incubation resulted in conversion of the enzyme to the fully oxidized, all ferric form of the enzyme 3þ 3þ (b3þ 558 b595 d ). This form of the enzyme was rapidly mixed in the stopped flow with an equal volume of 200 lM ubiquinone-1 + 6 mM DTT or, alternatively, 1 mM TMPD + 20 mM ascorbate, in order to monitor the rate of reduction of the all-ferric form of the enzyme. A strongly lipophilic tetrachlorobenzoquinone is expected to be an antagonist of UQH2-1 ubiquinol. Indeed, turnover of cytochrome bd with 100 lM UQH2-1 as electron donor in the presence of 16 lM tetrachlorobenzoquinone was decreased by 30–40% (to about 600–800 e s1). There was no effect of tetrachlorobenzoquinone on the ascorbate + TMPD-oxidase activity of the enzyme. 2.9. Data analysis The data were first analyzed with the use of Pro-Kineticist software package ‘‘ProK for PC’’ (Applied Photophysics) and selected data were imported into Origin 7 (Microcal) for further analysis and preparation of the figures.
3. Results Fig. 2A compares the kinetics of reduction of heme d in the oxoferryl and all-ferric forms of cytochrome bd. From the spectra/time surface collected, the kinetics at a wavelength pair 628 nm and 719 nm (DA628–719) was extracted to monitor the reduction of heme d. The kinetics of the reduction of the allferric form of the enzyme by UQH2-1 is extremely slow and non-homogeneous, reaching only about 25% of the expected maximum in 2 s, the maximal observation period in the experiment shown. This is in contrast to the extremely rapid reduction of heme d in the all-ferric enzyme (k 3400 s1),
Fig. 2. Kinetics of reduction of heme d in the oxoferryl and the allferric forms of cytochrome bd. Panel A. Reduction by UQH2-1: heme d in the oxoferryl form is reduced within about 10 ms, compatible with the turnover of the enzyme. No significant reduction of heme d in the all-ferric enzyme is observed in the same time range. Panel B. Reduction by TMPD: the reduction of the all-ferric form is significantly slower than the reduction of heme d in the oxoferryl form of the enzyme. Heme d in the oxoferryl form is reduced by TMPD much more slowly than with UQH2-1, see Section 2 for details.
reported in the pulsed radiolysis experiments of Kobayashi et al. [14]. Reduction of hemes b558 and b595 in the same experiment was even slower that reduction of heme d, with either DTT + ubiquinol or ascorbate + TMPD as electron donors (data not included) and took about 60 s for completion, whereas heme d reduction was complete in 20–30 s. Hence, it is likely that it is electron entry into the enzyme (reduction of b558), rather than intramolecular electron transfer, that limits the rate of reduction of ferric heme d in the all-ferric enzyme. In order to examine the reactivity of the oxoferryl state of cytochrome bd, this form of the enzyme was generated in situ in the stopped flow mixing chamber by oxidation of the fully reduced cytochrome bd with stoichiometric amount of O2 2þ 2þ 2þ 2þ b2þ þ O2 ¢ b2þ 558 b595 d 558 b595 d AO2
ð2Þ
2 2þ 2þ 3þ 3þ 4þ 2Hþ þ ðb2þ 558 b595 d AO2 Þ ! ðb558 b595 d @O Þ þ H2 O
ð3Þ
Formation of the oxycomplex (reaction 2) and subsequent oxidation of the reduced cytochrome bd by bound oxygen (reaction 3) are complete within the mixing time, in agreement with the known rate constants. At 2.5 lM oxygen (final con-
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centration after mixing), the expected rate for the reaction with O2 is about 2 · 109 M1 s1 · 2.5 lM O2 = 5 · 103 s1, s 200 ls (see [20]), and the oxidation yields largely the oxoferryl state of the enzyme (at least 80%). This was most clearly seen in the spectra from the experiments in which reduced TMPD was used as the reductant (data not shown), since the subsequent reduction of the enzyme in this case is much slower than with UQH2-1. The oxoferryl form of the enzyme is reduced quite rapidly by UQH2-1, and the reaction is completed in about 10 ms (Fig. 2A). During this time, up to 12 time-resolved spectra could be collected. The major part of the transiently oxidized cytochrome bd is actually reduced within the dead time of the mixing apparatus and only the tail end of this rapid reduction could be monitored with the current instrumentation. Global analysis of the spectra/time surface for the resolved part of the reduction process gave a rate constant of 200– 300 s1, which is close to enzyme turnover rate (1000 electrons s1) when multiplied by four electrons required to convert enzyme from the oxoferryl to the all ferrous state. The difference spectrum of the rapid phase of the reduction, as resolved by global analysis, is shown in Fig. 3. The spectrum shows clearly simultaneous disappearance of the oxoferryl state (trough around 670–680 nm) and appearance of reduced heme d (peak at 630 nm), as well as reduced heme b558 and heme b595 (the peaks at 595, 560 and 530 nm in the visible, and an asymmetric Soret band with a maximum at 431 nm and a shoulder around 438 nm). These data imply that the reaction monitored involves reduction of the enzyme by two equivalents of UQH2-1, since four electrons are required to 2þ 2þ generate the fully reduced form of the enzyme (b2þ 558 b595 d ) 2 3þ 3þ 4þ from the oxoferryl state (b558 b595 d @O ) . The first equivalent of UQH2-1 produces a partially reduced form of the en3þ 2þ zyme (b3þ 558 b595 d ), so these results imply that the rate of reduction by the second equivalent of UQH2-1 of the partially 3þ 2þ reduced enzyme (b3þ 558 b595 d ) is about as fast as the reaction of 2 3þ 4þ the oxoferryl species (b3þ 558 b595 d @O ) with the first equivalent of UQH2-1.
K. Yang et al. / FEBS Letters 582 (2008) 3705–3709
The reduction kinetics of the enzyme with a single electron donor TMPD reduced by excess ascorbate was also examined (Fig. 2B). Reduction of the all-ferric enzyme in this case is also very slow. As in the case of ubiquinol, the reaction of reduced TMPD with the oxoferryl form is more rapid than with the allferric form of the enzyme, although it is still much slower than observed with UQH2-1. The reduction of the all-ferric cytochrome bd by reduced TMPD occurs on a time scale of 0.1– 0.5 s, not in milliseconds. It cannot be excluded that the reaction of cytochrome bd with reduced TMPD is rate-limited by the oxidation of TMPD, i.e., TMPD is a much poorer electron donor than UQH2-1.
4. Discussion The data clearly show that the reduction of heme d in the oxidized (all-ferric) form of cytochrome bd by its natural two-electron substrate, ubiquinol, is much too slow to be part of the catalytic cycle. In contrast, the reaction of the oxoferryl form of cytochrome bd with ubiquinol is rapid and results in formation of the fully reduced enzyme. Presumably, the reaction proceeds in two sequential two-electron steps which are not time-resolved under the conditions of our experiments at a UQH2-1 concentration of 100 lM (the final concentration after mixing) 2 3þ 4þ UQH2 -1 þ ðb3þ 558 b595 d @O Þ ¢ UQ-1 þ H2 O 2þ 2þ þ ðb2þ 558 b595 d Þ
UQH2 -1 þ
3þ 2þ ðb3þ 558 b595 d Þ
¢ UQ-1 þ
2þ 2þ ðb2þ 558 b595 d Þ
ð4Þ ð5Þ
The rate of reaction 5 must be at least as fast as reaction 4 under the conditions of these experiments, since the appearance of reduced heme d and the reduced hemes b occur in the same time window. However, in the presence of excess of O2, the very rapid rate of binding of O2 with the one-electron reduced enzyme (microseconds) is expected to result in formation of the one-electron ferrous–oxy complex 3þ 2þ 3þ 3þ 2þ ðb3þ 558 b595 d Þ þ O2 ¢ ðb558 b595 d AO2 Þ
ð6Þ
Hence, when the concentration of O2 is high, reaction 6 would prevail, followed by reaction 3þ 2þ 2þ 2þ 2þ ðb3þ 558 b595 d AO2 Þ þ UQH2 -1 ¢ ðb558 b595 d AO2 Þ
Fig. 3. Difference spectrum of the rapidly reduced component of cytochrome bd in the reaction of the oxoferryl form reduced by UQH21 found by global analysis of the spectra/time surface. The spectrum is that of major product of the reaction formed in the first 50 ms minus the initial spectrum, which is primarily the oxoferryl form of the enzyme. The conditions correspond to the trace shown in Fig. 2A.
ð7Þ
The product of reaction 7 has sufficient electrons to split the O–O bond (reaction 3), provided protons are available. Under microaerophilic growth conditions, in which cytochrome bd is normally expressed in E. coli [21], the reactions 3þ 2þ of the one-electron reduced enzyme (b3þ 558 b595 d ) with O2 (reaction 6) and with UQH2-1 (reaction 5) might occur in the reverse order, which, however, makes no difference to the final outcome of the reaction cycle. It is interesting that the heme 3þ 2þ d ferrous–oxy complex (b3þ 558 b595 d AO2 ) is very stable, being the major form of the enzyme as it is isolated [22]. The dissociation of oxygen in the form of superoxide (O 2 ), which would be deleterious to the cell, is negligible. On the other hand, twoelectron reduction of the all-ferric cytochrome bd by UQH2 and its subsequent oxidation by oxygen could give rise to an unstable peroxide adduct of the ferric enzyme that could dissociate releasing hydrogen peroxide, or produce hydroxyl radicals in case of homolytic scission of the O–O bond. Hence, the intermediate states of cytochrome bd with an odd number
K. Yang et al. / FEBS Letters 582 (2008) 3705–3709
of electrons relative to the all-ferric form appear to be preferred in the catalytic cycle of the enzyme. These catalytic intermediate forms contain +3, +1 and 1 electrons relative to the all-ferric enzyme: (a) +3 electrons = all-ferrous þ2 þ2 þ3 þ3 þ2 ðbþ2 558 b595 d Þ; (b) +1 electron = oxycomplex ðb558 b595 d AO2 Þ; 2 þ3 þ4 b d @O Þ. (c) 1 = oxoferryl ðbþ3 558 595 The expectation from the previous work of Ju¨nemann et al. [12] and of Matsumoto et al. [13], based on steady-state kinetics, that the all-ferric form of cytochrome bd is not part of the catalytic cycle of the enzyme is directly verified by pre-steadystate kinetics. It remains to be seen why the one-electron reductant formed during the pulsed radiolysis experiments by Kobayashi et al. [14] reacts with the all-ferric enzyme rapidly. The explanation might be simply that the N-methylnicotinamide radical and solvated electron generated under the conditions of pulse radiolysis are much stronger reductants than the one-electron donor used in the current work, TMPD (E0m ¼ 260 mV) as well as than the ubiquinol/ubisemiquinone couple that serves as an initial reductant for cytochrome bd during the reaction of the enzyme with ubiquinol. One might also note, that the all-ferric forms of cytochrome bd were obtained in this work and in Ref. [14] by different methods, but it is highly unlikely that a variance in the preparations can account for the 1000-fold difference in reactivity of heme d. The observation that UQH2-1 is a poor reductant for the allferric form of the enzyme, but a good reductant for the oxofer2 3þ 4þ ryl ðb3þ 558 b595 d AO Þ and one-electron reduced enzyme 3þ 3þ 2þ (b558 b595 d ) suggests that the redox state of heme d plays a role in determining the rate of reduction of heme b558 (and, perhaps, of heme b595 as well). Further work is needed to clarify the mechanism of this effect. Recent work has shown that the redox state of heme b558 and heme b595 regulates the accessibility of heme d by O2 [23], thus, reciprocal regulation of the reactivity of the two hemes b by the redox and/or ligand-binding state of heme d may be a reasonable speculation. Evidently, the interactions between the three hemes play a critical role in determining the sequence of reactions in the catalytic cycle. Acknowledgements: This work was supported by grants from the National Institutes of Health (HL16101 to R.B.G.), the Wenner–Gren Foundations (to R.B.G.), the Civilian Research and Development Fund (CRDF Grant RUB1-2836-MO-06 to R.B.G. and A.A.K.), the Howard Hughes Medical Institute (International Scholar Award 55005615 to A.A.K.) and the Russian Foundation for Basic Research Grant 08-04-00093 (to V.B.B.)
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The fully oxidized form of the cytochrome bd quinol oxidase from E. coli does not participate in the catalytic cycle: Direct evidence from rapid kinetics studies Ke Yanga, Vitaliy B. Borisovb, Alexander A. Konstantinovb, Robert B. Gennisa,* b
a Department of Biochemistry, University of Illinois, 600, Southgoodwin Avenue, Urbana, IL 61801, USA A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russia
Received 28 July 2008; revised 13 September 2008; accepted 16 September 2008 Available online 26 September 2008 Edited by Miguel De la Rosa
Abstract Cytochrome bd catalyzes the two-electron oxidation of either ubiquinol or menaquinol and the four-electron reduction of O2 to H2O. In the current work, the rates of reduction of the fully oxidized and oxoferryl forms of the enzyme by the 2-electron donor ubiquinol-1 and single electron donor N,N,N 0 ,N 0 -tetramethyl-p-phenylendiamine (TMPD) have been examined by stopped-flow techniques. Reduction of the all-ferric form of the enzyme is 1000-fold slower than required for a step in the catalytic cycle, whereas the observed rates of reduction of the oxoferryl and singly-reduced forms of the cytochrome are consistent with the catalytic turnover. The data support models of the catalytic cycle which do not include the fully oxidized form of the enzyme as an intermediate. 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Cytochrome bd; Ubiquinol; Respiration
1. Introduction The cytochrome bd quinol oxidase is one of the two terminal oxidases in the respiratory chain of E. coli, and catalyzes the 2electron oxidation of either menaquinol or ubiquinol and the four-electron reduction of oxygen to water [1–5]. This enzyme is a member of a large family of respiratory oxidases that are found in many prokaryotes, both bacteria and archaea. There is no eukaryotic homologue, and the presence of bd-type oxidases in a number of human pathogens makes this enzyme a possible drug target. Cytochrome bd is a transmembrane heterodimer, with one copy each of two proteins coded by the genes CydA (subunit I; 58 kDa) and CydB (subunit II; 43 kDa) [6]. There is no sequence homology between cytochrome bd and the large superfamily of the heme–copper respiratory oxidases [7], and the bd-type oxidases do not contain copper [6]. During turnover, cytochrome bd does not pump protons but it generates a proton motive force due to the fact that the protons used in the reaction to make H2O come from * Corresponding author. Fax: +1 217 244 3186. E-mail address: [email protected] (R.B. Gennis).
Abbreviations: TMPD, N,N,N 0 ,N 0 -tetramethyl-p-phenylendiamine; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; UQH21, ubiquinol-1
the bacterial cytoplasm, whereas the protons from the oxidation of QH2, which is oxidized near the opposite side of the membrane, are released to the periplasm [8,9]. Cytochrome bd contains three heme prosthetic groups to transfer electrons from quinol to oxygen: heme b558, heme b595 and heme d. The low spin heme b558 is believed to be the entry site of electrons from quinol and is located near the putative quinol binding site. Heme d is the site where oxygen binds and is reduced to water. The role of the high spin heme b595 is ˚ from the high still not clear, but heme b595 is located about 10 A spin heme d [10] and minimally its role is to provide an electron to facilitate the reduction of oxygen bound to heme d. The membrane potential appears to be generated essentially by proton uptake accompanying the electron transfer from heme b558 to the oxygenated heme b595/heme d center, which generates a product with a high affinity for protons [8]. The isolated cytochrome bd appears to have a single binding site for quinols. Unlike the heme–copper oxidases that utilize ubiquinol as a substrate, such as cytochrome bo3 from E. coli, there is no evidence for a tightly bound quinone which does not readily exchange with the quinone pool in the membrane. Hence, the isolated cytochrome bd has no bound quinone and the fully reduced form of the enzyme contains three electrons for redox chemistry, corresponding to the ferrous forms of each of the three hemes. Four different states of heme d have been characterized spectroscopically in cytochrome bd. These are (1) ferric (Fe3+); (2) ferrous (Fe2+); (3) ferrous–oxy complex (Fe2+–O2) and (4) oxoferryl (Fe4+‚O2). A putative peroxy complex (supposedly, Fe3+–OOH) has been observed recently as a transient intermediate during oxidation of the fully reduced enzyme by O2 [11], but its structure requires further confirmation. Each of the other four forms of heme d can be generated as stable or metastable forms that can be examined and characterized. These forms suggest a sequence of events at heme d as O2 is converted to water Fe2þ ! Fe2þ O2 þO2
Fe4þ @O2
!
þe þHþ
!
Fe3þ OAOH
þe þ2Hþ H2 O
!
þe þHþ H2 O
Fe3þ ! Fe2þ þe
ð1Þ
The analysis of enzyme turnover, however, must take into account the redox status of the other two heme prosthetic groups, heme b558 and heme b595, which can exist in either the Fe2+ or Fe3+ states, and also the fact the ubiquinol is a two-electron donor.
0014-5793/$34.00 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.09.038
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The purpose of the current work is to directly test the proposal based on the steady-state kinetics studies [12,13] that 3þ 3þ the fully oxidized (b3þ 558 b595 d ) form of cytochrome bd may not be part of the catalytic cycle (Fig. 1). The model shown in Fig. 1 implies that the rate of reduction of the all-ferric form of the enzyme should be slow. Contrary to this proposal, fast reduction of heme d in the all-ferric enzyme was observed in pulse radiolysis studies [14]. The generation of a strong one-electron reductant in this experiment resulted in very rapid reduction of heme b558, equilibration within 10 ls between heme b558 and heme b595, followed by electron transfer to heme d with the rate constants of about 3400 s1 and 590 s1, for the ferric and the oxoferryl forms, respectively. The one-electron transfer to heme d in the ferrous–oxy form (Fe2+–O2) was about 100-fold slower than observed with the ferric and oxoferryl forms. Taken at face value, these data suggest that the all-ferric form should be reconsidered as a possible intermediate during steady state analysis, and raises questions about the catalytic competence of the ferrous–oxy complex as an intermediate. An important difference between the steady-state turnover and pulse radiolysis experiments is that the former normally utilizes a two-electron donor whereas the latter utilizes a one-electron reductant. This was pointed out by Kobayashi et al. [14], in reference to the slow single-electron transfer to the ferrous–oxy complex. In the current work, a rapid mixing/diode array spectrophotometer was used to monitor the rate of reaction of ubiquinol1 (UQH2-1) and of ascorbate-reduced N,N,N 0 ,N 0 -tetramethylp-phenylendiamine (TMPD) with the all-ferric form of cytochrome bd. The kinetics of these reactions were compared with the reactions with the oxoferryl form of the enzyme generated by transient oxidation of the fully-reduced cytochrome bd by a stoichiometric amount of oxygen. The results reveal very slow (seconds) reduction of the all-ferric enzyme by ubiquinol-1, whereas reduction of the oxoferryl form of cytochrome bd is at least
K. Yang et al. / FEBS Letters 582 (2008) 3705–3709
1000-fold more rapid and compatible with the turnover number of the enzyme. With a single-electron donor TMPD, the oxoferryl form of cytochrome bd is also a much better electron acceptor than the all-ferric form, however, the difference is much less pronounced than in the case of two-electron reductant UQH2-1. The data show directly that the all-ferric form of the enzyme is not likely to be part of the normal catalytic cycle.
2. Materials and methods 2.1. Strains and plasmids E. coli strain GO105 (cyd AB::kan, cyo, recA), which lacks both the cytochrome bo3 and cytochrome bd quinol oxidases [15], was used as the host strain for overexpressing the wild type cytochrome bd from plasmid pTK1 [15] which was introduced into the strain. 2.2. Cell growth and enzyme purification Large scale cell growth was carried out in 24 2 L flasks shaking at 220 rpm and 37 C using an Innova 4330 incubator shaker (New Brunswick Scientific) [16]. Cytochrome bd was purified as described previously [17]. The pooled fractions were concentrated using an Amicon concentrator with a 50 kDa molecular weight cut-off filter and then dialyzed three times against 50 mM sodium phosphate buffer, pH 7.8, containing 5 mM ethylenediaminetetraacetic acid (EDTA), 0.05% N-lauroylsarcosine. 2.3. Heme analysis The heme b content of purified cytochrome bd was measured by the pyridine hemochromogen assay, using an extinction coefficient for the wavelength pair 556.5540 nm, De556.5–540 = 24 mM1 cm1 [18]. The heme d content and enzyme concentration were determined from the absolute spectrum of the fully reduced enzyme, using the extinction coefficient De628–670 = 25 mM1 cm1 [8]. 2.4. Ubiquinol-1 and TMPD oxidase activity assays The steady state oxidase activity assays were performed as described previously by Zhang et al. [17]. The purified enzyme had a turnover number of about 1100 s1 with 100 lM UQH2-1 + 3 mM dithiothreitol (DTT) and about 160 s1 with 10 mM ascorbate + 0.5 mM TMPD.
Fig. 1. Catalytic cycle of ubiquinol-oxidase activity of cytochrome bd. The scheme is based on the work of Ju¨nemann et al. [12] as modified by Matsumoto et al. [13].
K. Yang et al. / FEBS Letters 582 (2008) 3705–3709
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2.5. UV–visible spectroscopic measurements Static absorbtion spectra were obtained with a UV-2101PC spectrophotometer (Shimadzu). 2.6. Stopped-flow experiments The stopped-flow experiments were performed at 20 C using an Applied Photophysics stopped flow with a photodiode array detector. The flow system was flushed thoroughly with argon-saturated buffer (50 mM sodium phosphate, 5 mM EDTA and 0.05% N-lauroylsarcosine, pH 7.8) before the experiments. The whole system was kept anaerobic by flowing argon during the experiments. The mixing ratio of the two solutions was 1:1. After mixing, 1600 spectra were collected in the 300–1200 nm range over a period ranging from 2 s to 1 min. At least three runs were performed for each time scale. The concentrations reported are the initial concentrations before mixing, if not stated otherwise. 2.7. Generation of the oxoferryl form of the enzyme Five micromolar of enzyme was fully reduced by 200 lM ubiquinone-1 + 6 mM dithiothreitol or by 1 mM TMPD + 20 mM ascorbate in an argon-saturated buffer (50 mM sodium phosphate, 5 mM EDTA and 0.05% N-lauroylsarcosine, pH 7.8). The fully reduced enzyme 2þ 2þ (b2þ 558 b595 d ) was then rapidly mixed in the stopped-flow apparatus with an equal volume of a solution containing approximately 5 lM O2 in the same buffer. The reduced cytochrome bd was rapidly oxidized by O2 yielding essentially the oxoferryl form of the enzyme 3þ 4þ 2 (b3þ 558 b595 d @O ), which was subsequently reduced by the excess reductant present in the solution. The oxidation reaction is too fast to resolve, but the tail end of the reduction kinetics could be observed spectroscopically. Spectroscopic analysis of the product of the reaction of O2 with the reduced enzyme showed that it contained approximately 80% oxoferryl and 20% ferrous–oxy complex (not shown). 2.8. Experiments with the all-ferric form of cytochrome bd Five micromolar of the ‘‘as isolated’’ cytochrome bd, mainly present 3þ 2þ as ferrous–oxy complex b3þ 558 b595 d AO2 , was incubated for 20 min with 16 lM tetrachlorobenzoquinone, a lipophilic strong oxidant [19], in argon-saturated buffer (50 mM sodium phosphate, 5 mM EDTA and 0.05% N-lauroylsarcosine, pH 7.8). The incubation resulted in conversion of the enzyme to the fully oxidized, all ferric form of the enzyme 3þ 3þ (b3þ 558 b595 d ). This form of the enzyme was rapidly mixed in the stopped flow with an equal volume of 200 lM ubiquinone-1 + 6 mM DTT or, alternatively, 1 mM TMPD + 20 mM ascorbate, in order to monitor the rate of reduction of the all-ferric form of the enzyme. A strongly lipophilic tetrachlorobenzoquinone is expected to be an antagonist of UQH2-1 ubiquinol. Indeed, turnover of cytochrome bd with 100 lM UQH2-1 as electron donor in the presence of 16 lM tetrachlorobenzoquinone was decreased by 30–40% (to about 600–800 e s1). There was no effect of tetrachlorobenzoquinone on the ascorbate + TMPD-oxidase activity of the enzyme. 2.9. Data analysis The data were first analyzed with the use of Pro-Kineticist software package ‘‘ProK for PC’’ (Applied Photophysics) and selected data were imported into Origin 7 (Microcal) for further analysis and preparation of the figures.
3. Results Fig. 2A compares the kinetics of reduction of heme d in the oxoferryl and all-ferric forms of cytochrome bd. From the spectra/time surface collected, the kinetics at a wavelength pair 628 nm and 719 nm (DA628–719) was extracted to monitor the reduction of heme d. The kinetics of the reduction of the allferric form of the enzyme by UQH2-1 is extremely slow and non-homogeneous, reaching only about 25% of the expected maximum in 2 s, the maximal observation period in the experiment shown. This is in contrast to the extremely rapid reduction of heme d in the all-ferric enzyme (k 3400 s1),
Fig. 2. Kinetics of reduction of heme d in the oxoferryl and the allferric forms of cytochrome bd. Panel A. Reduction by UQH2-1: heme d in the oxoferryl form is reduced within about 10 ms, compatible with the turnover of the enzyme. No significant reduction of heme d in the all-ferric enzyme is observed in the same time range. Panel B. Reduction by TMPD: the reduction of the all-ferric form is significantly slower than the reduction of heme d in the oxoferryl form of the enzyme. Heme d in the oxoferryl form is reduced by TMPD much more slowly than with UQH2-1, see Section 2 for details.
reported in the pulsed radiolysis experiments of Kobayashi et al. [14]. Reduction of hemes b558 and b595 in the same experiment was even slower that reduction of heme d, with either DTT + ubiquinol or ascorbate + TMPD as electron donors (data not included) and took about 60 s for completion, whereas heme d reduction was complete in 20–30 s. Hence, it is likely that it is electron entry into the enzyme (reduction of b558), rather than intramolecular electron transfer, that limits the rate of reduction of ferric heme d in the all-ferric enzyme. In order to examine the reactivity of the oxoferryl state of cytochrome bd, this form of the enzyme was generated in situ in the stopped flow mixing chamber by oxidation of the fully reduced cytochrome bd with stoichiometric amount of O2 2þ 2þ 2þ 2þ b2þ þ O2 ¢ b2þ 558 b595 d 558 b595 d AO2
ð2Þ
2 2þ 2þ 3þ 3þ 4þ 2Hþ þ ðb2þ 558 b595 d AO2 Þ ! ðb558 b595 d @O Þ þ H2 O
ð3Þ
Formation of the oxycomplex (reaction 2) and subsequent oxidation of the reduced cytochrome bd by bound oxygen (reaction 3) are complete within the mixing time, in agreement with the known rate constants. At 2.5 lM oxygen (final con-
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centration after mixing), the expected rate for the reaction with O2 is about 2 · 109 M1 s1 · 2.5 lM O2 = 5 · 103 s1, s 200 ls (see [20]), and the oxidation yields largely the oxoferryl state of the enzyme (at least 80%). This was most clearly seen in the spectra from the experiments in which reduced TMPD was used as the reductant (data not shown), since the subsequent reduction of the enzyme in this case is much slower than with UQH2-1. The oxoferryl form of the enzyme is reduced quite rapidly by UQH2-1, and the reaction is completed in about 10 ms (Fig. 2A). During this time, up to 12 time-resolved spectra could be collected. The major part of the transiently oxidized cytochrome bd is actually reduced within the dead time of the mixing apparatus and only the tail end of this rapid reduction could be monitored with the current instrumentation. Global analysis of the spectra/time surface for the resolved part of the reduction process gave a rate constant of 200– 300 s1, which is close to enzyme turnover rate (1000 electrons s1) when multiplied by four electrons required to convert enzyme from the oxoferryl to the all ferrous state. The difference spectrum of the rapid phase of the reduction, as resolved by global analysis, is shown in Fig. 3. The spectrum shows clearly simultaneous disappearance of the oxoferryl state (trough around 670–680 nm) and appearance of reduced heme d (peak at 630 nm), as well as reduced heme b558 and heme b595 (the peaks at 595, 560 and 530 nm in the visible, and an asymmetric Soret band with a maximum at 431 nm and a shoulder around 438 nm). These data imply that the reaction monitored involves reduction of the enzyme by two equivalents of UQH2-1, since four electrons are required to 2þ 2þ generate the fully reduced form of the enzyme (b2þ 558 b595 d ) 2 3þ 3þ 4þ from the oxoferryl state (b558 b595 d @O ) . The first equivalent of UQH2-1 produces a partially reduced form of the en3þ 2þ zyme (b3þ 558 b595 d ), so these results imply that the rate of reduction by the second equivalent of UQH2-1 of the partially 3þ 2þ reduced enzyme (b3þ 558 b595 d ) is about as fast as the reaction of 2 3þ 4þ the oxoferryl species (b3þ 558 b595 d @O ) with the first equivalent of UQH2-1.
K. Yang et al. / FEBS Letters 582 (2008) 3705–3709
The reduction kinetics of the enzyme with a single electron donor TMPD reduced by excess ascorbate was also examined (Fig. 2B). Reduction of the all-ferric enzyme in this case is also very slow. As in the case of ubiquinol, the reaction of reduced TMPD with the oxoferryl form is more rapid than with the allferric form of the enzyme, although it is still much slower than observed with UQH2-1. The reduction of the all-ferric cytochrome bd by reduced TMPD occurs on a time scale of 0.1– 0.5 s, not in milliseconds. It cannot be excluded that the reaction of cytochrome bd with reduced TMPD is rate-limited by the oxidation of TMPD, i.e., TMPD is a much poorer electron donor than UQH2-1.
4. Discussion The data clearly show that the reduction of heme d in the oxidized (all-ferric) form of cytochrome bd by its natural two-electron substrate, ubiquinol, is much too slow to be part of the catalytic cycle. In contrast, the reaction of the oxoferryl form of cytochrome bd with ubiquinol is rapid and results in formation of the fully reduced enzyme. Presumably, the reaction proceeds in two sequential two-electron steps which are not time-resolved under the conditions of our experiments at a UQH2-1 concentration of 100 lM (the final concentration after mixing) 2 3þ 4þ UQH2 -1 þ ðb3þ 558 b595 d @O Þ ¢ UQ-1 þ H2 O 2þ 2þ þ ðb2þ 558 b595 d Þ
UQH2 -1 þ
3þ 2þ ðb3þ 558 b595 d Þ
¢ UQ-1 þ
2þ 2þ ðb2þ 558 b595 d Þ
ð4Þ ð5Þ
The rate of reaction 5 must be at least as fast as reaction 4 under the conditions of these experiments, since the appearance of reduced heme d and the reduced hemes b occur in the same time window. However, in the presence of excess of O2, the very rapid rate of binding of O2 with the one-electron reduced enzyme (microseconds) is expected to result in formation of the one-electron ferrous–oxy complex 3þ 2þ 3þ 3þ 2þ ðb3þ 558 b595 d Þ þ O2 ¢ ðb558 b595 d AO2 Þ
ð6Þ
Hence, when the concentration of O2 is high, reaction 6 would prevail, followed by reaction 3þ 2þ 2þ 2þ 2þ ðb3þ 558 b595 d AO2 Þ þ UQH2 -1 ¢ ðb558 b595 d AO2 Þ
Fig. 3. Difference spectrum of the rapidly reduced component of cytochrome bd in the reaction of the oxoferryl form reduced by UQH21 found by global analysis of the spectra/time surface. The spectrum is that of major product of the reaction formed in the first 50 ms minus the initial spectrum, which is primarily the oxoferryl form of the enzyme. The conditions correspond to the trace shown in Fig. 2A.
ð7Þ
The product of reaction 7 has sufficient electrons to split the O–O bond (reaction 3), provided protons are available. Under microaerophilic growth conditions, in which cytochrome bd is normally expressed in E. coli [21], the reactions 3þ 2þ of the one-electron reduced enzyme (b3þ 558 b595 d ) with O2 (reaction 6) and with UQH2-1 (reaction 5) might occur in the reverse order, which, however, makes no difference to the final outcome of the reaction cycle. It is interesting that the heme 3þ 2þ d ferrous–oxy complex (b3þ 558 b595 d AO2 ) is very stable, being the major form of the enzyme as it is isolated [22]. The dissociation of oxygen in the form of superoxide (O 2 ), which would be deleterious to the cell, is negligible. On the other hand, twoelectron reduction of the all-ferric cytochrome bd by UQH2 and its subsequent oxidation by oxygen could give rise to an unstable peroxide adduct of the ferric enzyme that could dissociate releasing hydrogen peroxide, or produce hydroxyl radicals in case of homolytic scission of the O–O bond. Hence, the intermediate states of cytochrome bd with an odd number
K. Yang et al. / FEBS Letters 582 (2008) 3705–3709
of electrons relative to the all-ferric form appear to be preferred in the catalytic cycle of the enzyme. These catalytic intermediate forms contain +3, +1 and 1 electrons relative to the all-ferric enzyme: (a) +3 electrons = all-ferrous þ2 þ2 þ3 þ3 þ2 ðbþ2 558 b595 d Þ; (b) +1 electron = oxycomplex ðb558 b595 d AO2 Þ; 2 þ3 þ4 b d @O Þ. (c) 1 = oxoferryl ðbþ3 558 595 The expectation from the previous work of Ju¨nemann et al. [12] and of Matsumoto et al. [13], based on steady-state kinetics, that the all-ferric form of cytochrome bd is not part of the catalytic cycle of the enzyme is directly verified by pre-steadystate kinetics. It remains to be seen why the one-electron reductant formed during the pulsed radiolysis experiments by Kobayashi et al. [14] reacts with the all-ferric enzyme rapidly. The explanation might be simply that the N-methylnicotinamide radical and solvated electron generated under the conditions of pulse radiolysis are much stronger reductants than the one-electron donor used in the current work, TMPD (E0m ¼ 260 mV) as well as than the ubiquinol/ubisemiquinone couple that serves as an initial reductant for cytochrome bd during the reaction of the enzyme with ubiquinol. One might also note, that the all-ferric forms of cytochrome bd were obtained in this work and in Ref. [14] by different methods, but it is highly unlikely that a variance in the preparations can account for the 1000-fold difference in reactivity of heme d. The observation that UQH2-1 is a poor reductant for the allferric form of the enzyme, but a good reductant for the oxofer2 3þ 4þ ryl ðb3þ 558 b595 d AO Þ and one-electron reduced enzyme 3þ 3þ 2þ (b558 b595 d ) suggests that the redox state of heme d plays a role in determining the rate of reduction of heme b558 (and, perhaps, of heme b595 as well). Further work is needed to clarify the mechanism of this effect. Recent work has shown that the redox state of heme b558 and heme b595 regulates the accessibility of heme d by O2 [23], thus, reciprocal regulation of the reactivity of the two hemes b by the redox and/or ligand-binding state of heme d may be a reasonable speculation. Evidently, the interactions between the three hemes play a critical role in determining the sequence of reactions in the catalytic cycle. Acknowledgements: This work was supported by grants from the National Institutes of Health (HL16101 to R.B.G.), the Wenner–Gren Foundations (to R.B.G.), the Civilian Research and Development Fund (CRDF Grant RUB1-2836-MO-06 to R.B.G. and A.A.K.), the Howard Hughes Medical Institute (International Scholar Award 55005615 to A.A.K.) and the Russian Foundation for Basic Research Grant 08-04-00093 (to V.B.B.)
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[7]
[8]
[9] [10]
[11]
[12]
[13]
[14] [15]
[16]
[17]
[18] [19]
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