effect of the Tyr-143 -- Phe mutation is a change in the rate-determining step in the ... type enzyme the main rate-determining step is proton abstraction at the C-2 ...
187
Biochem. J. (1992) 285, 187-192 (Printed in Great Britain)
Tyr-143 facilitates interdomain electron transfer in flavocytochrome b2 Caroline S. MILES,* Nathalie ROUVIERE-FOURMY,t Florence LEDERER,t F. Scott MATHEWS,$ Graeme A. REID,§ Michael T. BLACK§ and Stephen K. CHAPMAN* 1 * Edinburgh Centre for Molecular Recognition, Department of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, Scotland, U.K., t CNRS URA 1461, Hopital Necker, 161 rue de Sevres, 75743 Paris Cedex 15, France, t Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, U.S.A., and § Edinburgh Centre for Molecular Recognition, Institute of Cell and Molecular Biology, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, Scotland, U.K.
The role of Tyr- 143 in the catalytic cycle of flavocytochrome b2 (L-lactate: cytochrome c oxidoreductase) has been examined by replacement of this residue with phenylalanine. The electron-transfer steps in wild-type and mutant flavocytochromes b2 have been investigated by using steady-state and stopped-flow kinetic methods. The most significant effect of the Tyr-143 -- Phe mutation is a change in the rate-determining step in the reduction of the enzyme. For wildtype enzyme the main rate-determining step is proton abstraction at the C-2 position of lactate, as shown by the 2H kineticisotope effect. However, for the mutant enzyme it is clear that the slowest step is interdomain electron transfer between the FMN and haem prosthetic groups. In fact, the rate of haem reduction by lactate, as determined by the stopped-flow method, is decreased by more than 20-fold, from 445 + 50 s-I (25 °C, pH 7.5) in the wild-type enzyme to 21 + 2 s-' in the mutant enzyme. Decreases in kinetic-isotope effects seen with [2-2H]lactate for mutant enzyme compared with wild-type, both for flavin reduction (from 8.1 + 1.4 to 4.3 0.8) and for haem reduction (from 6.3 + 1.2 to 1.6 + 0.5) also provide support for a change in the nature of the rate-determining step. Other kinetic parameters determined by stopped-flow methods and w-ith two external electron acceptors (cytochrome c and ferricyanide) under steady-state conditions are all consistent with this mutation having a dramatic effect on interdomain electron transfer. We conclude that Tyr-143, an active-site residue which lies between the flavodehydrogenase and cytochrome domains of flavocytochrome b2, plays a key role in facilitating electron transfer between FMN and haem groups.
INTRODUCTION
MATERIALS AND METHODS
Flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase, EC 1.1.2.3) from baker's yeast (Saccharomyces cerevisiae) is a soluble component of the mitochondrial intermembrane space [1], where it catalyses the oxidation of L-lactate to pyruvate and transfers electrons to cytochrome c [2]. The enzyme is a tetramer of identical subunits of Mr 57 500 [3], with each subunit composed of two functionally distinct domains: a flavodehydrogenase domain containing flavin mononucleotide (FMN) and a cytochrome domain containing protohaem IX [2]. The crystal structure of flavocytochrome b2 has been solved to 0.24 nm resolution [4] and shows two crystallographically distinguishable subunits in the asymmetric unit. In subunit 1, both the cytochrome and flavodehydrogenase domains are clearly seen in the electron-density map. However, in subunit 2 there is a molecule of pyruvate, the reaction product, located at the active site of the flavodehydrogenase domain, and the cytochrome domain is not visible, owing to positional disorder. The threedimensional structure also reveals that, in subunit 1, the side chain of Tyr- 143 is hydrogen-bonded to a haem propionate, whereas in subunit 2 this residue forms a hydrogen bond with the carboxylate group of pyruvate (Fig. 1). From the structure, then, it would appear that Tyr-143 is uniquely placed to play an important, and perhaps key, role in controlling electron transfer from substrate to flavin and from flavin to haem. In the present paper we describe work in which we have probed the role of this residue by constructing and characterizing a mutant form of the enzyme (Y143F), in which Tyr-143 has been replaced by Phe.
DNA manipulation, strains, media and growth
1 To whom correspondence should be addressed.
Vol. 285
Escherichia coli MM294 was used for expression of wild-type and mutant flavocytochromes b2 as reported elsewhere [5]. Sitedirected mutagenesis was performed as previously described [6] using the oligonucleotide 295A (GTGGGCCTTCTATTCCT) (Oswel DNA Service, University of Edinburgh, Edinburgh, Scotland, U.K.). Standard methods for growth of E. coli, plasmid purification, DNA manipulation and transformation were performed as described in Maniatis et al. [7]. Enzymes Wild-type and Y143F flavocytochromes b2 were isolated from frozen E. coli cells using the purification procedure previously reported [5]. All stopped-flow experiments were carried out with enzyme purified by hydroxyapatite chromatography as described elsewhere [5]. Steady-state experiments were carried out initially on enzyme purified to the (NH4)2SO4 precipitation step and subsequently verified with hydroxyapatite-purified enzyme [5]. Purified enzyme preparations were stored under nitrogen at 4 °C as precipitates in 70% satd. (NH4)2S04. Flavocytochrome b2 concentrations were determined spectrophotometrically by using previously published molar absorption coefficients [8]. Kinetic analysis All kinetic experiments were carried out at 25.0 + 0.1 °C in Tris/HCl at pH 7.5 and I0.10. The buffer concentration was 10 mm in HCl, with I adjusted to 0.10 M by addition of NaCl. Steady-state kinetic measurements involving the enzymic oxi-
188
C. S. Miles and others was monitored at 420 nm as previously described [8]. Cytochrome
(a)
c reduction was monitored at 550 nm, published molar absorption coefficients for oxidized and reduced forms of the protein [9] being used. Stopped-flow measurements were carried out with an Applied Photophysics SF. 17MV microvolume stopped-flow spectrophotometer with a dead time of A 1 ms. Flavin reduction was monitored at 438.3 nm (a haem isosbestic point), and haem reduction was monitored at 557 nm. Analysis of stopped-flow data was performed using the SF. 17MV spectrometer software, which allows non-linear regression analysis of traces to analytical equations. Traces were fitted to single or double exponentials as appropriate; for wild-type and mutant enzymes, single exponential fits were sufficient at low lactate concentrations. At least four runs were performed at each L-lactate concentration. Km and kcat parameters were determined using the SF. 17MV spectrometer software, either by directly analysing experimental data using non-linear regression analysis or by analysing the appropriate double-reciprocal plots using linear-regression analysis; both methods gave self-consistent results. Kinetic parameters reported here are those from non-linear regression analysis. Kinetic-isotope effects were measured by using [2-2H]lactate prepared and purified as previously described [10]. The purity of the [2-2H]lactate was confirmed by 'H-n.m.r. spectrometry.
(b)
RESULTS
Y143
E OH
Fig. 1. Structure of the active sites in the crystallographically distinguishable subunits 14,111 (a) Subunit 1 (pyruvate absent); (b) subunit 2 (pyruvate present). In subunit 1, Tyr-143 can be clearly seen hydrogen-bonding to a haem propionate group and in subunit 2 to the carboxylate group of pyruvate. W664 is a water molecule, not tryptophan. R376 is arginine-376 (etc.).
dation of L-lactate were performed using Pye-Unicam SP. 8-400 and Beckman DU62 spectrophotometers. Ferricyanide and cytochrome c (horse heart; Type VI; Sigma) were used as electron acceptors where appropriate and at the concentrations indicated in Tables 1 and 3 (below). Reduction of ferricyanide
Steady-state kinetic parameters The results of steady-state kinetic measurements with L-[21H]lactate and L-[2-2H]lactate as substrates and with ferricyanide and cytochrome c as electron acceptors are presented in Table 1. It is clear that there are a number of significant differences between the kinetic properties of wild-type and Y143F enzymes. The kcat. value for Y143F enzyme is dramatically reduced with cytochrome c as electron acceptor; however, with ferricyanide as electron acceptor the kcat value is essentially the same, within experimental error, as that for wild-type flavocytochrome b2. The fact that these differences in kcat values are dependent on the nature of the electron acceptor must reflect differences in the electron flow to these acceptors. The 2H kinetic-isotope effect measured under steady-state conditions for wild-type enzyme with ferricyanide as electron acceptor (4.7 + 0.4) is identical, within experimental error, with the previously published kinetic-isotope-effect value of 5 [10] (which was determined under somewhat different conditions). This value showed that the major rate-limiting step is proton abstraction at C-2 of lactate [10]. This is illustrated in Scheme 1,
Table 1. Steady-state kinetic parameters and 2H-kinetic-isotope effects (KIE) for wild-type and Y143F flavocytochromes b2 All experiments were carried out at 25 °C in Tris/HCl buffer, pH 7.5 (IO.10). Acceptors were used at the following concentrations: [cytochrome c], 30 /M (this is 75 % saturating for wild-type enzyme and 95 % saturating for Y143F enzyme); [ferricyanide], 1 mm for wild-type enzyme (> 90 % saturating) and 8 mm for Y143F enzyme (71 % saturating). kcat is expressed in electrons transferred/s per mol of enzyme (since L-lactate is a twoelectron donor, these values can be halved to express them in terms of mol of substrate reduced/s). Abbreviations: ['H]Lac, L-[2-'H]lactate; [2H]Lac, L-[2-2H]lactate; WT, wild-type enzyme; Y143F, Tyr-143 -. Phe mutant enzyme.
Enzyme WT Y143F
kcat. (S-')
Km (mM)
Electron acceptor
['H]Lac
[2H]Lac
['H]Lac
[2H]Lac
Ferricyanide Cytochrome c Ferricyanide Cytochrome c
400+10 207+ 10 400+30 22+2
86+ 5 70+10 195 +20 13+2
0.49+0.05 0.24+0.04 2.90+0.23 0.23 +0.03
0.76+0.06 0.48+0.10 5.14+1.10 0.73 +0.03
10-5 Xkeat./Km (M-'l-s') ['H]Lac [2H]Lac 8.2 8.6 1.4 1.0
1.1 1.5 0.4 0.2
KIE 4.7+0.4 3.0+0.6 2.0+0.4 1.7+0.5
1992
Interdomain electron transfer in flavocytochrome b2 H373
(a) R376
189 Table 2. Values of
K. for the electron acceptors ferricyanide and cytochrome c All experiments were carried out at 25 °C in Tris/HCl buffer, pH 7.5 (I 0.10). The L-lactate concentration was kept at 10 mm throughout (this is 95 % saturating for wild-type enzyme and 78 % saturating for Y143F enzyme).
K349
(DH3N 0t~\
\--
R
o
D282
I~
Km Electron
Y143
Enzyme *~
Y254
~_OH
WT Y143F
(b)
H373
K349
(DH3N
