8023
J. Phys. Chem. 1992,96,8023-8027
Photoinitiated Electron Transfer between Cytochrome c and Cytochrome c Oxidase Using a Novel Uroporphyrin/NADH Reducing Systemt Randy W. Larsen,t Jay R. Winkler,%and Sunney I. Chan*vt Arthur Amos Noyes Laboratory of Chemical Physics and The Beckman Institute, California Institute of Technology. Pasadena, California 91I25 (Received: December 19, 1991; In Final Form: April 20, 1992)
We have employed a novel photoreduction system to investigate the electron-transfer reaction between cytochrome c and cytochrome c oxidase. In this system, the photogenerated uroporphyrin triplet state is quenched through electron transfer to ferricytochrome c. The corresponding uroporphyrin r a t i o n radical is rapidly reduced by nicotinamide adenine dinucleotide (NADH) resulting in the in situ generation of ferrocytochrome c. In the presence of cytochrome c oxidase, cytochrome c is reoxidized biphasically while the corresponding reduction of cytochrome a appears to be monophasic. In addition, the fast-phase rate constants are dependent upon the concentration of cytochrome c oxidase giving a first-order intracomplex electron-transfer rate constant, ( k d ,of 1829 248 s-’. The ratio of electrons transferred from ferrocytochmmec to cytochrome a is 1:l indicating that cytochrome a is the ultimate acceptor of the electrons.
*
Introduction Cytochrome c oxidase (CcO) is an integral membrane protein which catalyzes the oxidation of ferrocytochrome c and the corresponding reduction of dioxygen to water. The catalytic function of CcO is carried out using four redox active metal centers. These centers consist of two heme A chromophores and two Cu ions.’.* The reduction of dioxygen takes place at a binuclear cluster consisting of one heme A chromophore (designated cytochrome a3)and one Cu ion (designated Cue). The two remaining metal centers (designated cytochrome a and CuA) have reduction potentials near that of cytochrome c during turnover and mediate the electron flow from ferrocytochrome c to the binuclear cluster. Since the electrons from. ferrocytochrome c originate in the cytosol of the mitochondrion and the protons that are consumed in the formation of water are taken from the inner matrix, the redox free energy between ferrocytochrome c and molecular oxygen is converted, in part, into a transmembrane protonmotive force during respiration. In addition, concurrent with this electron flow, protons can be vectorially pumped across the inner membrane from the matrix to the cytosol to augment the transmembrane electrochemical gradient. The reaction between CcO and its physiological redox partner, cytochrome c, is of specific interest since the elucidation of the electron-transfer pathway@) from cytochrome c to the dioxygen reduction site may provide relevant information concerning the mechanism of redox-linked proton translocation in CcO. Previous studies of this reaction have primarily involved the use of stopped-flowtechniques which have demonstrated that the reaction between cytochrome c and CcO is ionic strength dependent and multiphasic with rates that decrease with increasing ionic ~trength.~”A serious drawback in these studies is the inability to monitor the rapid kinetic processes occurring within the mixing time of the stopped-flow in~trument.~” In order to investigate this reaction on a faster time scale, several groups have developed reduction systems in which cytochrome c is reduced in situ. Recently, Hazzard et a1.6 investigated the rate of electron input into CcO using the photogenerated semiquinone of 5-deazariboflavin. These studies were extended by Pan et al.’ to include electron transfer between cytochrome c and a CuA-depletedderivative ofrCcO. Using an alternative method, Millet and Durhams examined the rate of electron transfer between several Ru-polypyridine derivatives of cytochrome c and CcO in which cytochrome c is reduced in situ via excited-state Author to whom correspondence should be addressed. ‘Contribution No. 8550 from the Arthur Amos Noyes Laboratory of Chemical Physics, California Institute of Technology, Pasadena CA. This work is supported by Grant GM22432 from the National Institute of General Medical Sciences, U. S. Public Health Service. *Arthur Amos Noyts Laboratory of Chemical Physics. $The Beckman institute.
quenching of Ru(I1) by the heme Fe of ferricytochrome c. In this work, we describe yet another technique for the in situ generation of reduced cytochrome c using photwnsitized fnabasc uroporphyrin. These studies are based upon previous observations by Zhou et a1.9 and Cho et al.1° who demonstrated that the reversible photoinitiated electron transfer between water soluble porphyrins and cytochrome c occurs via porphyrin triplet-state quenching by ferricytochrome c according to the following mechanism:
-- ++
P IP* Ip* + 3p*
3P* + cytochrome c(3+)
(1)
(2)
P+ cytochrome c(2+) (3)
P+ + cytochrome c(2+)
P cytochrome c(3+) (4) where P refers to the free-base porphyrin. A major drawback to the use of this system for studying the electron-transfer reaction between cytochrome c and CcO is the rapid thermal intramolecular back-reaction (reaction 4), which is sufficiently fast (k4 1 X 108 M-’ sd lo) to compete with the correspondingelectron transfer between the two protein complexes. In the presence of an a p propriate porphyrin radical scavenger, however, reaction 4 can be significantly inhibited. We demonstrate that reduced nicotinamide adenine dinucleotide (NADH) rapidly quenches the ?r-cationradical of uroporphyrin thus inhibiting reaction 4. The inhibition of reaction 4 allows for the study of electron transfer between cytochrome c and CcO via in situ reduction of CytOchKnne c by free-base uroporphyrin.
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Maiterials and Methods Uroporphyrin I dihydrochloride (Porphyrin Products, Ogden, UT), NADH (Sigma), and horse heart cytochrome c ( S iType VI) were used without further purification. Cytochrome c oxidase was isolated and purified by the method of Hartzell and Beinert” and stored at -80 OC until needed. Stock solutions of uroporphyrin (1.5 mM), NADH (100 mM), and cytochrome c (1.5 mM) were prepared in 100 mM HEPES buffer, 0.1% Brij-35, pH 7.4. In the case of the uroporphyrin, the buffer solution was first brought to pH 9.5 by the addition of 0.2 M KOH to ensure monomerization of the porphyrin. The stock solutions were prepared in vials with septum cap and purged with a stream of Ar for 30 min. Samples for transient absorption measurements were prepared as follows: 2.98 mL of 100 mM HEPES containing 15 pM uroporphyrin was brought to pH 7.4 and placed in a l c m quartz optical cell with a septum cap. The solution was then degassed for 30 min by flushing with Ar. Cytochrome c and NADH were then added to a final concentration of 15 W M for cytochrome c and 1 mM for NADH using a gas-tight Hamilton syringe. Ab-
0022-36S4/92/2096-8023$03.00/0@ 1992 American Chemical Society
Larsen et al.
8024 The Journal of Physical Chemistry, Vol. 96, No. 20, 1992 0.20
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Wavelength ( n m ) Figure 1. Steady-state difference spectrum of a solution containing 10 pM cytochrome c, 10 pM cytochrome c oxidase, 10 pM uroporphyrin, and 1 mM NADH in 100 mM HEPES, 0.1% Brij-35, pH 7 . 4 (a) after exposure to white light for 30 min and (b) with incubation in the dark for 30 min. The control sample contained 10 pM cytochrome c, 10 pM cytochrome c oxidase,and 10 p M uroporphyrin with no NADH. Spectra were obtained in a 1-cm path-length quartz cuvette.
sorption transients were then colkected at 550 nm to monitor the extent of cytochrome c reduction; In experiments with cytochrome c oxidase, the enzyme was added to a final concentration of 15 pM from a 200 pM stock solution. Final solution ionic strength was 60 mM. Absorption transients were then recorded at 550 and 605 nm. All transients were the average of 10 laser shots obtained at a repetition rate of 10 Hz. The data were fitted using a nonlinear least squares algorithm with Enzfitter software. The photoreduction of cytochrome c was accomplished by exciting the uroporphyrin at 500 nm using the output of a Lambda Physik FL 3002 dye laser (Coumarin 480 dye) pumped by a Lambda Physik LPX21Oi excimer laser. Excitation pulse energies were -3.5 mJ/pulse (20-ns pulse width). Single wavelength transient absorption kinetics were obtained using a 75-Wxenon arc lamp as a probe Source. The signal was passed through an Instruments SA 1680B double monochromator (2-nm band-pass) placed after the sample and detected with a photomultiplier tube. The transient signal was amplified using either a 200-MHz quasi difftrential amplifer (for kinetics out to 750 M) or a LeCroy DSP 1402E programmable amplifier (for kinetics out to 10 ms) and digitized using a Tektronix R7 10 200-MHz transient digitizer interfaced to a 386-based microcomputer.
Results Figure 1 displays the effects of steady-state illumination upon a solution containing 16 pM cytochrome c, 10 pM CcO, 10 pM uroparphyrin, and 1 mM NADH. Under steady-state illumination with a white light source (tungsten lamp) for 20 min, both cytochrome c and CcO become fully reduced. This is apparent by the appearance of positive bands at 550 and 605 nm in the illuminated minus control difference spectrum (Figure 1, spectrum a). The control spectrum is obtained from a sample containing 10 pM cytochrome c, 10 pM CcO, and 10 pM uroporphyrin in the absence of NADH. Figure 1, spectrum b displays the corresponding dark minus control differencespectrum. In this case, no visible reduction of either cytochrome c or CcO takes place, indicating that the reduction is a photoinitiated process. The transient reduction of cytochrome c by uroporphyrin is dsplayed in Figure 2. In the absence of NADH (Figure 2, trace a), cytochrome c is rapidly reduced through direct quenching of the uroporphyrin triplet state. This rapid reduction is followed by a slower oxidation of cytochrome c through back-electron transfer in which the correspondinguroporphyrin *-cation radical is d u d . In the presence of NADH (Figure 2, trace b), however, the back-electron-transfer reaction is significantly inhibited. Figwe 3A displays the 5 5 0 . ~ transient 1 of a solution containing 15 rM cytochrome c, 15 pM uroporphyrin, and 1 mM NADH in the absence (Figure 3A, trace a) and presence (Figure 3A, trace
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Time (ms)
Figure 2. Absorption transient obtained at 550 nm of a solution of 15 cytochrome c and 15 pM uroporphyrin in the absence (a) and presence (b) of 1 mM NADH. Buffer conditions are the same as those in Figure 1.
pM
b) of 15 pM CcO. The corresponding double-exponential fit of the data in Figure 3A, trace b is shown in Figure 3B. The presence of CcO in the solution induces a biphasic oxidation of cytochrome c with an observed pseudo-first-order rate constant of 1344 f 8 1 s-l for the fast phase and 52 i 23 s-l for the slow phase. The corresponding transient reduction of CcO is shown in Figure 4A. The reduction of the low-pokntial cytochrome a (Figure 4A, trace a), monitored at 605 nm (absorbance maximum for reduced cytochrome a ) , occurs with an observed pseudo-first-order rate constant of 1151 f 30 s-I. The single-exponential best fit to the data is shown in Figure 4B. To ensure that the observed reduction of cytochrome a is associated with electron transfer from cytochrome c and not through direct quenching of the uroporphyrin by CcO, the transient absorption spectra of cytochrome a (monitored at 605 nm) w a obtained ~ for a solution containing 15 pM CcO, 15 pM uroporphyrin, and 1 mM NADH (Figure 4A, trace b). In this case, no absorbance change could be observed indicating that the observed reduction of cytochrome a is due to electron transfer from cytochrome c. One possible concern in the use of the uroporphyrin/ NADH/cytochrome c system to photoinitiate electron transfer to CcO is the effect of electrostatic interactions between the negatively charged uroporphyrin and the positively charged cytochrome c. Transient complex formation between cytochrome c and CcO could interfere with the triplet-state quenching of uroporphyrin by cytochrome c since the same charged domain of the cytochrome c involved in transient binding of cytochrome c to CcO may also be involved in the transient binding and subsequent quenching of the trjplet-state uroporphyrin. To clarify this point, we have compared the photoinduced reduction of cytochrome c by triplet-state quenching of uroporphyrin in the absence and presence of CcO (Figure SA, traces a and b, respectively). The singleexponential best fits to the data are shown in Figure 5B, C, respectively (the reduction of cytochrome c by uroporphyrin triplet-state quenching is pseudo first order since the overall yield of porphyrin triplet state (
