13089
J. Phys. Chem. 1993,97, 13089-13091
Electron Transfer from Cytochrome bg to Methemoglobin Tiecheng Qiao, Jennifer Simmons, Doris AM Horn, Robin Chandler, and George McLendon' Department of Chemistry University of Rochester, Rochester, New York I4627 Received: September 3, 1993; In Final Form: October 9, 1993'
Electron transfer in the hemoglobin reductase (cytochrome bs) hemoglobin system has been studied as a function of driving force, temperature, ionic strength, and pH. The driving force dependence is consistent with a Marcus' theory analysis where h = 0.9 V. However, the apparent temperature dependence is larger than expected from such an analysis. Analysis of the data suggests that the physiological reaction may proceed via a simple bimolecular collisional mechanism, without preassociation of the protein in a stable complex.
Introduction Electron-transfer reactions between proteins play major roles in metabolism, ranging from energy transduction in mitochondria to biological "repair" mechanisms.' A key example of the latter is provided by the methemoglobin reductase system in erythrocytes. Daily, about 3% of hemoglobin is oxidized to (inactive) Fe(II1) methemoglobin? This hemoglobinis restored to the active Fe(I1) form via an enzymatic reduction system, in which direct electron transfer to methemoglobin occurs from the redox protein cytochrome bS. The bs:Hb system has been broadly investigated by several groups.2 In particular, both direct kinetic and linked enzymatic studies have provided estimates of the protein-protein binding constants and rate constants for the reduction of methemoglobin by bs.2 Direct measurements of bS:Hb complex formation were independently reported by Mauk et ala3 Such binding, which is generally believed to precede electron transfer, was shown to be sensitive to pH, ionic strength, and temperature. In subsequent work, Mauk and Poulos4 suggested a structural model for this complex. Some features of this model have been questioned (e.g., the heme-heme distance predicted by this model is shorter than that estimated by energy tran~fer).~ It does explain other key observations, including mutational effects, and serves as a good starting point for further refined models. Most recently, we have begun systematic dynamic and structural studies of this system using a variety of approaches. In this paper, we explore some of the key features which control the electron-transfer rate between these proteins. Following in the footsteps of Closs and Miller, the Franck-Condon term is explored by varying either reaction free energy or temperature. As an approach to further understanding the details of electron coupling between the (bS) donor and (Hb) acceptor, the donor acceptor distance has been estimated by (Forster) energy transfer. Measurements were performed under conditions in which the rate constant is modulated by ionic strength or pH.
TABLE I: Excited-State Decay Rates for MHbbs Complex (M = Hi,Mg,ZnP HzHb/bs MgHb/bs Zn Hb/bs a
9.9 X lo2
N.A.
1.4 X lo3 2.7 X lo3
1.4 X lo2 3.1 X lo2
NA = 0.9
= 0.7
Conditions 1 mM Kphos buffer at pH 6.2 ( b = 1.4 mM).
Zinc hemoglobin was prepared as previously described.5b The reconstitution of wild type cyt bs with deuteroporphyrin was done by a similar heme exchange procedure.' Photochemical reactions were performed on a transient spectrophotometer. A halogen lamp was used as a probe to measure background absorbance of the Hb/cyt bS sample at 460 nm. A frequency-doubled Nd:YAG laser (Quanta-Ray) was used in the Q-switched mode to excite the photoactive Hb at 532 nm. The absorbance at 551 nm was monitored as a function of time using a monochrometer and a photomultiplier tube. The thermal chemical reactions were measured using a stopflow UV-vis spectrophotometer (Kin Tek). The absorbance at 577 nm was monitored as a function of time after the mixing of methemoglobin with reduced cyt bs. The temperature was controlled by a built-in temperature control unit.
Results and Discussion Dependence of Rate on Reaction Exothermicity. Just as for small molecules, the reorganization energy is a key determinant of electron transfer rates for proteinsa8 Following Marcus, the reorganization energy can be experimentally estimated by systematically varying the reaction free energy. At pH 7, the rate constant for the reaction Fe(I1) bs Fe(II1) Hb is relatively slow (AG= 0.1 V, k,, 1 s-l). In preliminary work, we showed that this rate of intracomplexelectron transfer can be significantly increased by increasing the reaction free energy.5 For example, photoinduced electron transfer from photoexcited 3(Zn hemoglobin)* to Fe(II1) bs occurs with AG 0.9 V and k,, 1600 s-1 (fitting the data to a single exponential). Such differences are consistent with a maximal reaction rate when AG A 2 0.9 V. Since this initial report, more detailed studies have shown that the apparent first-order kinetics for the photochemical reaction are better described by double-exponentialdecay processes. Values for these double exponentials are compiled in Table I. We note that the kinetics can be further complicated by a process (presumably energy transfer) which can depend on the incident laser intensity. This complication was eliminated by working at lower intensities, so that the observed rates were intensity independent. Assuming a constant value of A for both the photochemicaland thermal reactions, Marcus theory then predicts the rate for the Fe(1I) bs/Fe(III) Hb system (AG = 0.1 V) as k,, = 1600 e~p-(O.9~.1)2/~(0.9)~~ = 1.6 s-1 in reasonably good
-
+
=
=
Experimental Section Human Hb was prepared following the procedure of Antonini with minor modifications.sb Methemoglobin was obtained by oxidizing oxyhemoglobin with K3[Fe(CN)a] and run through G-25-150size exclusion column to eliminate the oxidizing agent. Methemoglobin concentration was determined by using the peak at 405 nm with extinction coefficient 179 mM-I cm-1. Wild type rat cyt bS was purified according to the method of Beck et aL6 Cyt b5 was reduced by using spectrometrictitration with Na2S204. The concentration of cyt bS was determined by using the peak at 412.5 nm with extinction coefficient 130 mM-l cm-l. Both proteins used in this work were purified to the purity index greater than 5.0. *Abstract published in Advance ACS Absrracrs, November 15, 1993.
0022-365419312097-13089$04.00/0 0 1993 American Chemical Society
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Qiao et al.
13090 The Journal of Physical Chemistry, Vol. 97, No. 50, 1993 I
I
0.4 ~
15
~
1.1
a.4
S.6
6.0
4.8
TIME (seconds) Ngure 1. Stopflow kinetics of wild type rat cyt b5 (1 5 fiM) with human methemoglobin (60 pM)at 25 OC, 10 mM phosphate buffer, pH 6.20.
,"
Y
0
d
I
2Y 3
Y
1
1
V '
0.0
0.4
0.8
1.2
-AG Figure 2. Plot of electron-transfer rate constant (log kc,)versus reaction free energy (AG). Experiments were run at 25 OC, pH 6.20, 1 mM phosphate buffer. (1) FeIIb5/Fe111Hb, (2) 3(H2porphyrinHb)*/Fe1I1b5, (3) 3(ZnHb)*/Fe111b5,(4) 3( MgHb)*/Fe1I1b,.
I
/
1.2j
25
35
45
55
65
kbk,,[metHb]/l
+ [metHb]&.
the work of Mauk, we can estimate k,, = kob/[Hb]Kb = krpp/Kb z 2 X 104 M-1 s-'/104 = 2 s-l, at 10 mM phosphate buffer, pH 6.2. This appears to support a simple preequilibrium model, as previously reported. However, our own work involves slightly different conditions (phosphate buffer, not cacodylate buffer) as well as a different species (rat, not cow) and b5 oxidation state (Fe(I1) not Fe(II1)). Saturationconsistent with Mauk'sKbva1ues3 is not observed at 1 or 5 mM. At 5 mM, Mauk's value of Kb = 3 X lo4 M-1 (at 25 OC,pH 6.2) predicts rate saturation above [Hb] > 30 pM, but no such saturation is observed up to 80 pM. This requires Kb < lo4 M-I and ket > 5 s-I. Rate saturation is approached at both 1 and 5 mM phosphate buffer pH 6.2,lO O C . If such saturation is ascribed to complex formation, we obtain values of k,, = 2.6 s-I, Kb = 1.5 X lo4 M-1. This Kb value is approximately 10-foldlower than that estimated by Mauk. Since the extrapolated k,, values change dramatically for different ionic strengths, we conclude that a simple preequilibrium model does not hold rigorously across a range of ionic strengths. An earlier report5a suggested that a preequilibrium model did hold at 1-10 mM Pi. The limiting ket value appeared to be independent of ionic strength in contrast to other protein complexes (e.g., cytc: ccp) where k,, changes with ionic strength.9 This conclusion was based on assuming the validity of previous Kb values3 which are now shown to be invalid under our conditions. In fact, we must seriously consider the possibility that the thermal Fe111Hb:FeIIb~ reaction proceeds optimally by a second-order collisional mechanism, rather than via (preequilibrium) binding. This is explored in more detail in the following section on temperature dependence. Temperature Dependence of the Rate Constant. In principle, an independent approach to determiningthe reorganization energy can come from studies of the temperature dependence of the rate (in the "normal" region: AG
