The Site and Mechanism of Dioxygen Reduction in Bovine ... · inner mitochondrial membrane, which catalyzes the reaction: O2 + 4 cytochrome c2+ + 4 Hf (1) More than 90% of oxygen

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. Issue of September 25, pp. 13641-13654, 1988 Printed in U. S. A.

The Site and Mechanism of Dioxygen Reduction in Bovine Heart Cytochrome c Oxidase*

(Received for publication, December 14, 1987, and in revised form, March 17, 1988)

Olof EinarsdottirS, Miles G . ChocB, Sharon Weldonli, and Winslow S. Caugheyll From the Department of Biochemistry, Colorado State University, Fort Collins, Colorado 80523

The site and mechanism of dioxygen reduction in cytochrome c oxidase from bovine heart muscle have been investigated. The rate of cytochrome c2+ oxidation by O2 is shown to be affected by several factors: 1) pH, with optima at 5.65 and 6.0, 2) temperature between 0 and 29 "C, with E, = 13 kcal mol", 3) D2O exchange, with a reduction in rate of 50% or more at the pH optima, and 4) the addition of ethylene glycol or glycerol, which significantly lowers the rate. The extremely narrow (Auto - 4 cm") infrared stretch bands at -1964 and -1959 cm" for liganded CO are only slightly affected by factors 1-4 or by changes in the oxidation state of metals other than the heme a3 iron. These results indicate a stable, unusually immobile O2 reduction site well-isolated from the external medium, a characteristic expected to be important for oxidase function. Precise stereochemical positioning of hydrogen donors adjacent to 0 2 liganded to heme a3 iron can be expected in order to achieve the optimization of the timetdistance relationships required for enzyme catalysis. These findings support a novel mechanism of 0 2 reduction via a hydroperoxide intermediate within a reaction pocket that experiences little change in conformation during the hydrogen and electron transfer steps.

Cytochrome c oxidase (CcO)' is a complex protein of the inner mitochondrial membrane, which catalyzes the reaction:

O2 + 4 cytochrome c2+ + 4 Hf (1)

More than 90% of oxygen consumption in biological systems

*This research was supported by United States Public Health Service Grant HL-15980. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: INC-4, Mail Stop C345, Los Alamos National Laboratories, Los Alamos, NM 87545.

07936. 3 Present address: Sandoz Pharmaceutical, East Hanover, NJ

7 Present address: Case Western Reserve University School of Medicine, Department of Biochemistry, Cleveland, OH 44106.

1) To whom correspondence should be addressed Dept. of Biochem- istry, Colorado State University, Ft. Collins, CO 80523.

oxidase; CcO(IV), CcO(III), CcO(II), CcO(I), and CcO(0) represent The abbreviations and trivial names used are: CcO, cytochrome c

cytochrome c oxidase at different oxidation states, the fully oxidized, 1-, 2-, 3-, and 4-electron-reduced species, respectively; CcO(O)CO, fully reduced carbonyl cytochrome c oxidase; CuL, the copper that binds external ligands and is most closely associated with FeL; Hb, hemoglobin; Mb, myoglobin; VCO, carbonyl stretch band wavenumber at maximum absorbance; FeL, the iron of the heme A that binds ligands (the heme a3).

+ 2 H20 + 4 cytochrome c3+

may involve this enzyme (1) which functions as a proton pump coupled to the electron transfers from cytochrome c to O2 (2, 3). CcO can also function as a carbon monoxide diox- ygenase catalyzing the reaction, 2 CO + O2 + 2 COZ (4).

The enzyme isolated from bovine heart muscle is a complex of up to 13 subunits (5, 6), phospholipids, two heme A chro- mophores (cytochromes u and us), coppers, zinc, and magne- sium (3, 7-10). The importance of the two hemes and two copper atoms in electron transfers from cytochrome c to 0 2 is well recognized, but roles for the other metals remain unclear. The stoichiometries of the metals support the existence of CcO as a dimer containing 5 Cu, 4 Fe, 2 Zn, and 2 Mg in the native state (8, 9). The metal contents and x-ray diffraction data for crystals of our oxidase preparation also support a dimeric structure (11) although the minimal unit for reduction of O2 by cytochrome c is apparently a monomer containing two hemes.

The complex oxidation-reduction system of cytochrome c oxidase has been studied by various spectroscopic techniques, with attention largely focused on electron transfers from cytochrome c to the O2 reduction site (3, 10). However, the details of the structure of the dioxygen reduction site per se and the chemical steps by which dioxygen is reduced to water within CcO remain unclear. Characterization of the 02-binding site is essential for understanding the mechanism of the dioxygen reduction to water and for elucidation of the protein structures that facilitate this reaction.

Infrared spectroscopy provides an effective and direct method to study the binding of ligands to hemeproteins, including CcO (12-14). Carbon monoxide is an infrared active probe of 02-binding sites because CO generally binds to the same sites that bind 0 2 . The frequencies and bandwidths of the CO infrared stretch bands are sensitive to the bonding and environment of the CO ligand. Therefore, these parameters can give valuable information on the ligand site structure (13). Cyanide infrared spectra indicate that CcO can bind cyanide at either cuprous or cupric copper and at either ferrous or ferric heme u3, depending upon the overall redox level of the enzyme (15). Present evidence indicates that only the heme u3 iron (FeL) and one Cu (Cu,) are accessible to external ligands in each monomer. CO binds to reduced heme u3 under physiological conditions and has been shown to bind to a reduced copper in mitochondrial preparations when CO is photodissociated from ferrous heme u3 at low temperatures (16). Previous infrared studies of CO binding to the ferrous heme u3 of CcO at different redox levels of the enzyme give clear evidence for two CO stretch bands of Gaussian shape (14). This suggests that two conformations of different structure exist at the ligand-binding site. Each band is extraordinarily narrow (AuH - 4 cm" compared to 8-20 cm" in other hemeproteins) (13, 17-19). The ligand environment at heme a3 remains essentially unaffected by changes in oxidation state at the other redox active metals as demonstrated by the

13641

13642 Mechanism of Dioxygen Reduction by Cytochrome c Oxidase

similar uco and AuH values for all redox levels that bind CO (14).

We report here further infrared and electronic spectral studies of CO binding to the O2 reaction site of CcO. The effects of temperature, pH, DzO, and organic cosolvents on carbonyl infrared and visible Soret spectra and on enzyme activity have been investigated. The information obtained on the structure of the ligand-binding site and the factors that influence oxidase activity provide new insight into the catalytic mechanism of 0 2 reduction to HzO.

MATERIALS AND METHODS

Bovine heart cytochrome c oxidase was isolated from fresh bovine heart as described earlier (9, 12). The final product was dialyzed against 10 mM sodium phosphate buffer, pH 7.4, and concentrated to -2 mM in terms of heme A under 20 psi N2 on Amicon XM-50 membranes. Upon further concentration (-36 h) a precipitate, which can be in the form of microcrystals ( l l ) , formed and was collected by centrifugation at 10,000 rpm for 15 min. The product was soluble in 10 mM phosphate buffer, pH 7.4, with no added detergent to give an enzyme solution >2.0 mM in heme A.

Horse heart cytochrome c (Sigma, Type VI) was used without further purification. "Sweetser" reagent, which was used to remove trace amounts of 0 2 from CO and NO, was prepared from proflavine, methylviologen, and EDTA from Sigma and/or Aldrich, and was activated by illumination (20). USP grade NZ (99.9%) and CP grade CO were obtained from General Air Service and Supply.

Sample Preparation-CcO is isolated in its fully oxidized (resting) form. To obtain the fully reduced enzyme, CcO(O), a solution of fully oxidized enzyme, CcO(IV), was deoxygenated for 1 h with N2 gas, which had been passed through an oxygen removal cannister (Dla- mond Tool and Die Co.) and two scrubbing bottles of Sweetser reagent to remove oxygen and moisten the gas. The deoxygenated solution was reduced under Nz with sodium dithionite. The fully reduced enzyme carbonyl, CcO(O)CO, was obtained by blowing 12C160 over the solution of fully reduced enzyme for 30-60 min. To obtain the 13C160 and 12C180 enzyme complexes, the dithionite-treated enzyme in a gas-tight Hamilton syringe was flushed repeatedly with the respective isotopically labeled CO. Formation of the carbonyl complex was confirmed by visible and infrared spectroscopy (14). The enzyme in D20 buffer was obtained from enzyme solution in Hz0 buffer by exchanges in an Amicon Diaflow apparatus.

The pH of the CcO(0)CO complex was adjusted by adding variable amounts of CO-saturated high ionic strength buffer, 0.6 M citrate, 0.6 M phosphate, or 0.6 M borate (brought to different pH values with 0.6 N HCl) to CcO(0)CO in 0.01 M phosphate, pH 7.4, or by dialysis against appropriate buffer solutions under a CO atmosphere.

Actiuity Measurements-Activities were measured spectrophoto- metrically following the oxidation of reduced horse heart cytochrome c at 550 nm as described by Smith (21). The cytochrome c was reduced with dithionite and passed through a Sephadex G-15 or G-25 column to remove excess sodium dithionite and its reaction products. The effects of pH on enzyme activity were examined in 0.1 M sodium phosphate buffers at 24 "C in either Hz0 or D2O over the pH range 4.5-8.5 and 5.0-7.5, respectively. The D20 buffers were prepared by dissolving appropriate amounts of NaH2P04 and Na2HP04 in D20. The enzyme assay reaction was initiated by adding 10 gl of 1 gM oxidase solution at pH 7.4 to 3 ml of buffer solution containing 15 PM reduced cytochrome c. The reaction was followed for 100-200 s. The rate constants (the slopes of the first order plots) were determined by logarithmic regression. Rates are expressed in units of s"/ mg of protein/3 ml assuming 10 nmol of heme A/mg of protein (9). Activity uersus ionic strength measurements were carried out at pH 6.0 (24 1 "C) in sodium phosphate buffers over the concentration range 0.018-0.2 M. The effects of addition of either ethylene glycol or glycerol (5,15, or 25%, v/v) were measured in 0.1 M sodium phosphate buffer, pH 6.0. The dependence of the rate of the oxidase reaction on temperature was observed in the presence and absence of added organic cosolvents over the range 6.5 to 37 "C.

Recording of Spectra-Visible and Soret spectra were recorded with a Cary 17 spectrophotometer. Enzyme concentration was determined using E605nm = 20 mM" cm" for the fully reduced enzyme in terms of heme iron (12). The visible spectra were examined before and after the infrared spectral measurements by placing the infrared cell di- rectly in the Cary 17 cell compartment. Infrared spectra were recorded

on a computer-interfaced (Tektronix 4051) Perkin-Elmer model 180 spectrophotometer in absorbance mode under dual beam operation at a scan rate of 11 cm"/min, a resolution of 1.8 cm" at peak maximum, 20-fold ordinate expansion, and a time constant of 4. Data points were collected every 0.1 cm". Both sample and reference cell were maintained at the same temperature in variable temperature cell holders as required to obtain a flat base line in the infrared spectra. The temperature was monitored with a copper-constantan thermocouple inserted in the cell window. The reference cell for the isotope and temperature measurements contained oxidized enzyme of identical concentration placed in a precisely matched CaFz cell (Beckman FH-01) of 0.053 mm pathlength. A variable pathlength CaFz cell (Perkin-Elmer) containing water was used as a reference cell for the pH studies and the samples dialyzed against aqueous: organic solvent mixtures. Eighteen to 24 single scans were accumu- lated for the isotope and temperature studies. The CO spectra obtained at various pH values are an average of 3-4 single scans from 2000-1920 cm".

qesiduol

-,-. ._ ... , , . . . . , . . . . I I I

1980 1960 1940 WAVENUMBER (cm")

FIG. 1. Carbonyl infrared spectra of 2.2 m M CcO(0)CO in 10 m M sodium phosphate buffer at pH 7.4 and 24 "C. Top spectrum, the infrared spectrum slope corrected to the base line shown. Middle spectrum, the top spectrum, two Gaussian curves with the band parameters listed for the CII and CIII bands in Table I, and the residual spectrum (the difference between the observed spectrum and the sum of the two Gaussian curves) with absorption on the right and left sides of the major absorption. Bottom spectrum, the top spectrum and four Gaussian curves using band parameters listed in Table I. Residual, the difference obtained upon subtraction of the sum of the four calculated curves shown in the bottom spectrum from the observed spectrum. The residual spectrum corresponds closely to the base line and demonstrates the success of the curve fitting procedure with four bands.

13643 Mechanism of Dioxygen Reduction by Cytochrome c Oxidase TABLE I

Band parameters from deconvolution of C-0 infrared spectra of fully reduced cytochrome c oxidase carbonyls The enzyme is - 2 mM in total heme A in H20 or DzO 10 mM sodium phosphate buffer, pH 7.4, at 24 "C.

CI CII CIII CIV Isotope/solvent

UCO AulI2 Area" uco Av1/2 Area" w o Awl/, Area" w o Aul12 Area" cm" k 0.4 cm" f 0.3 % f 0.5 cm" f 0.2 em" f 0.2 % f 0.8 cm" k 0.2 cm" k 0.2 % f 0.8 cm" f 0.4 cm" k 0.3 % & 0.5

'2C'60/Hz0 1955.2 4.0 3.1 1959.6 4.0 13.3 1963.5 4.2 79.1 1969.4 4.2 4.5 'ZC'60/D20 1955.7 4.0 2.2 1959.9 3.7 15.3 1963.3 4.0 79.2 1967.7 4.0 3.3 13C160/Hz0 1910.0 4.0 3.6 1914.9 4.0 13.1 1918.8 4.2 78.9 1924.4 4.2 '2C'SO/H~0 1911.5 4.0

4.4 3.2 1915.6 4.0 13.1 1919.4 4.0 79.0 1924.3 4.2 4.7

a The area is expressed in terms of the percent of total area of the sum of the areas of bands CI, CII, CHI, and CIV. The area for a given deconvoluted band of Gaussian shape was calculated from the relationship: area = 1.065 X (Au1/2) X (absorbance at U C O ) .

I I I I

BOVINE c c o . c o n 0.0051

HUMAN i \ 0.021 Hb.CO

1980 1960 1940 1920

RABBIT -

BOVINE M b.CO

1980 1940 1900 WAVENUMBER (ern")

FIG. 2. Carbonyl infrared spectra of CcO(O)CO, hemoglobin CO, and myoglobin CO. Top, bovine heart CcO(0)CO at pH 7.4 and 24 "C. Upper middle, human HbCO at pH 7.4 and 30 "C from Ref. 25. Lower middle, rabbit HbCO at neutral pH and 29 "C from Ref. 25. Bottom, bovine heart MbCO at pH 7.4 and 20 "C from Ref. 24. The spectra were deconvoluted into Gaussian curves using the band parameters listed in Table 11.

Analysis of Infrared Data-Optimization of signal-to-noise ratio was attained by using a high enzyme concentration (2.2-2.5 mM) combined with spectral accumulation and computer-assisted averag- ing. Slope correction and determination of the base line are necessary for successful deconvolution (curve fitting) of the spectrum.

An infrared band can be characterized by three parameters, frequency at maximum absorbance ( u in cm"), width at half the maximum absorbance (ALA,+ in cm"), and integrated area (B in m"' cm") (13). Determination of the infrared parameters for each contributing band was accomplished by applying a curve-fitting routine in which the height, frequency, bandwidth, and shape were chosen manually. The closeness of the fit of the chosen bands to the actual spectrum can be judged by the flatness of the residual, i.e. the difference spectrum, obtained by subtracting the sum of the theoretical curves from the observed spectrum. Better curve fitting was obtained using a pure Gaussian function than a combination of Gaussian and Lor- entzian line shapes. Although the ideal shape of a single absorption band is represented by a Lorentzian function (22), the CO stretch bands of myoglobin and hemoglobin have been satisfactorily deconvoluted using a Gaussian band shape (18, 19). This is in agreement with studies of Seshadri and Jones (23) who have shown that solvent- solute interactions, the existence of multiple conformers, and instru- mental conditions can give rise to Gaussian shapes.

RESULTS

12 16 Infrared Spectra of CcO(0)CO"The infrared spectrum of

C 0 bound to fully reduced CcO exhibits a major asymmet- ric absorbance (Fig. 1, top panel) which can be deconvoluted into two bands of Gaussian shape (Fig. 1, middle panel) (14). However, a curve calculated for only two bands deviates from the observed spectrum on the high and low frequency sides of the major absorbance. The sum of the four bands of Gaussian shape shown in the bottom panel of Fig. 1 corresponds closely to the observed spectrum. At room temperature the four computed bands appear at 1955.2, 1959.6, 1963.5, and 1969.4 cm-' with relative areas 1:4:24:1.4 and a bandwidth of -4 cm-' in each case (Table I). The four bands observed in the CO infrared spectra of hemoglobin and myoglobin carbonyls have much wider bandwidths and differ substantially in their relative intensities (Fig. 2 and Table 11) (17-19, 24, 25).

Spectra were also measured in D20 solutions. The exchange of D20 for H 2 0 was verified by comparison of the CcO(O)CO/ D20 infrared spectrum recorded over the 4000-1200 cm" region with previously reported spectra for hemeprotein carbonyls in D2O (13). Substitution of D20 for H20 had negligible effect on visible and Soret absorption spectra of CcO(1V) and CcO(0)CO (not shown). The frequencies of the two major CO infrared bands in the D20 spectrum are shifted 0.2-0.3 cm" compared to HzO (Table I). The shift of -0.2 cm" in the major deconvoluted peak (CIII, Table I) is clearly experimen- tally significant, while the shifts of the weaker peaks are within probable experimental error of the deconvolution procedure.

The effects of isotopic labeling of CO (13C160 and 12C180) on the CO stretch frequencies of the four CO bands were also

13644 Mechanism of Dioxygen Reduction by Cytochrome c Oxidase TABLE I1

C - 0 stretch band parameters from deconvolution of infrared spectra of hemeprotein carbonyls at different temperatures and pH values

Protein Temperature Band CI Band CII Band CIII Band CIV

uco Aulj2 Area" uc0 AvlIz Area" vco Au1/2 Area" uco Area" o c PH

em" cm" % cm" cm" % cm" cm-I % cm" cm-' % Bovine cytochrome c oxidase 24 7.4 1955.2 4.0 2.5 1959.6 4.0 13.6 1963.4 4.2 80.6 1969.6 4.2 3.2

4 7.4 1955.2 4.0 2.7 1959.7 3.8 10.5 1963.6 4.0 82.7 1968.2 4.2 4.1

Human Hbb

Rabbit Hbb

Bovine Mbd

30 9.5 1930 10 1.4 1942.5 8.5 2.9 1951.0 8.0 94.1 1969.0 9 1.6 30 7.4 1934 10 1.3 1943.5 7.0 3.3 1951.2 8.0 92.9 1969.0 9 2.5 4 7.4 e 1943 10.5 3.2 1950.9 7.8 96.0 1969.5 7.5 0.8

30 5.0 1930 10 1.1 1943 8 2.7 1951.5 7.9 87.6 1968.5 9 8.6

29 7 1929.1 10.5 20.1 1946.8 11 7.4 1951.8 7.9 70.2 1970 8 2.3 5 7 1928.7 10.2 20.6 1944 10 1.9 1951.2 7.8 75.6 1971 8 1.9

29 5 1930.4 10 16.8 1945 9 3.2 1951.2 7.9 68.0 1968 10 12.0

35 7.8 1938 18.5 51.3 1943.5 9.0 42.8 1953.5 9.0 2.9 1965 10 3.0 8 7.8 1938 17 45.4 1943.4 8.4 51.1 1953.6 8.0 2.5 1965 10 1.0

20 7.4 1937.8 18.3 48.7 1943.5 8.7 45.5 1953.8 9.0 2.9 1965 10 2.9 20 4.7 1938.8 18.5 29.4 1945.6 9.7 40.5 1955.2 10.5 7.0 1965.2 10.6 23.2

Percent of sum of area for the sum of areas of bands CI, CII, CIII, and CIV. Band area is calculated as given in Table I.

Data from Ref. 25. e No band detected.

Data from Refs. 18 and 24.

determined (Fig. 3). The isotope shifts are near those calculated for diatomic CO (Table 111). The small deviations from diatomic behavior are systematic in two respects. First, the substitution of 13C always results in an isotope shift greater than the diatomic value (by -1-2 cm", Table 111) while the substitution of "0 always results in a shift less than diatomic (by -3 cm"). This behavior is typical of isotopically substi- tuted metal carbonyls (26). Second, the isotope shifts for both 13C and "0 approach the diatomic value as frequencies increase.

Oxidation State Effects-The CO infrared spectrum for each of the four redox levels (0, I, 11, and 111) can be convoluted into four bands. The changes in the band parameters that occur as the oxidation level varies are small but significant (Table IV). The frequencies decrease from I11 to I1 to I to 0 and the intensity of the major band (CIII) is greater at the more reduced levels, 0 and I (Fig. 4 and Table IV).

Temperature Effects-Fig. 5 shows the effect of lowering the temperature from 24 to 4 "C on the infrared spectra of CcO(0)CO in Hz0 and DzO buffers. The spectral changes are minor and fully reversible (Table 11). A small but significant change in the relative intensities of the two major computed bands (CII and CIII) occurs. The temperature difference spectrum (4 minus 24 "C) in Fig. 5 contains a positive band at 1964 cm" and a negative band at 1960 cm". At 24 "C the area of the 1960 cm" band comprises 14% (15% in DZO) of the total area, but at 4 "C it is 11% (13% in DzO). The total integrated areas of all the bands are the same at the two temperatures. The relative intensity of the CO stretch bands observed for hemoglobin and myoglobin carbonyls are more sensitive to temperature (Table 11).

Increasing the temperature of the CcO(0)CO solution from 24 to 37 "C increased the intensity of the two minor bands at 1969 and 1955 cm-l, decreased the major conformer at 1963 cm", and a new band appeared at 1973 cm". The changes were similar in DzO and HzO. Bringing the sample back from 37 to 24 "C did not fully restore the spectrum originally observed at 24 "C. Keeping the enzyme at 37 "C resulted in a gradual broadening of the minor band at 1955 cm" and an

intensification of the new 1973 cm" band (Fig. 6). Increasing the temperature to 43 "C further enhanced the 1973 cm" band, a trend that continued with either incubation at 43 "C or increasing the temperature to 52 "C (Fig. 6). Eventually, the major band at 1963 cm" merged with the 1973 cm" band giving rise to a broad absorbance ( AvH - 25 cm") centered at 1970-1975 cm" (Fig. 6). Lowering the temperature back to 24 "C did not change the CO spectrum, suggesting that irreversible changes had occurred at the ligand-binding site.

The changes in the CO infrared spectra were accompanied by significant changes in the visible and Soret spectra (Fig. 7). No change was observed in the visible and Soret spectra of CcO(0)CO over 22 h at 24 "C and 10 h at 4 "C in either HzO or D20 (Fig. 7). Increasing the temperature to 37 "C resulted in a loss of intensity at 604 nm, and the shoulder at 592 nm became less prominent. Incubation at 37 "C and a subsequent increase to 43 "C gradually shifted the a band from 604 to 599 nm accompanied by a further decrease in intensity. The shoulder at 592 nm disappeared and the peaks at 550 and 518 nm lost intensity. The Soret peak shifted from 431 to 434 nm, and a shoulder which appeared at -420 nm intensified at high temperatures. The changes in the visible and Soret spectra at high temperatures are consistent with CcO no longer being fully saturated with CO and/or becoming partially oxidized (14).

pH Effects-Infrared and electronic spectra of the fully reduced carbonyl enzyme, CcO(O)CO, were recorded over the pH range 4.4 to 11.4. Between pH 5.4 and 10.8 the infrared spectra can be deconvoluted into two major bands at -1963 and -1959 cm" with no significant changes in vco or AVH (Table V). The slope-corrected spectra (Fig. 8) and the residuals of the deconvoluted spectra (not shown) indicate minor bands at -1969 and -1955 cm" at the various pH values. However, signal-to-noise ratios are inadequate to positively establish these minor bands. As the pH shifted from 5.4 to 9.8, the 1959 cm" band became more intense relative to the 1963 cm" band (Fig. 8 and Table V). Between pH 10 and 11 the intensification of the 1959 cm" band became increasingly pronounced. Changes in the relative areas of CO stretch bands

Mechanism of Dioxygen Reduction by Cytochrome c Oxidase 13645 I I 1

C ~ O ( O ) Y O -

0.005 ] -

I I I

Residual

I I I

1940 1920 1900

I 1, I

I

Residual

1

'C'"O - " ~ ' " 0 0.002 f

-

Y 1940 1920 I900

WAVENUMBER (cm") FIG. 3. Effect of isotopic substitution on carbonyl infrared

spectra of CcO(0)CO (2.2 mM) in 10 mM sodium phosphate at pH 7.4 and 2 4 "C. Top spectrum, observed spectrum of I3Cl6O and four Gaussian curves used in deconvolution. Upper residual, the difference between the top observed spectrum and the sum of the Gaussian curves. Middle spectrum, observed spectrum of 12C180 and four Gaussian curves used in deconvolution. Lower residual, the difference between the middle observed spectrum and the sum of the Gaussian curves. The parameters used for the Gaussian curves in the deconvolutions are listed in Table 111. The closeness of the residuals to the base line is a measure of the success of the deconvolution procedure. Bottom spectrum, difference spectrum for the middle observed spectrum minus the top observed spectrum.

are much greater for hemoglobin and myoglobin carbonyls between pH 5 and 10 (Table 11) (17, 24, 25). Below pH 5 or above pH 11, the infrared spectra of CcO(0)CO contain broad bands (AvH - 20 cm-') centered at -1973 and -1965 cm" at pH 4.4 and 11.4, respectively (Fig. 8). Returning the enzyme

solution to neutrality from either pH extreme did not change the infrared spectra. Therefore, an irreversible change in enzyme structure occurred at both pH extremes.

The visible and Soret spectra of CcO(0)CO exhibited no detectable change in the a (604 nm) or Soret (431 nm) bands over the pH range 5.4 to 10.3. Significant changes were observed above pH 10.3 and below 5.4. Above pH 11 the spectrum changed in 1 h from an (Y band at -602 nm and a Soret band at -431 nm to bands at -588 and -425 nm, whereas vco remained centered at -1965 cm-'. After the return of the solution to neutral pH, the visible spectrum with Amax at 420, 430 (shoulder), 536, 575, and 600 nm suggested that a modification of the heme A had occurred which may be similar to the borohydride-modified CO complex reported by Tzagoloff and Wharton (27). Upon acidification to below pH 5.4, the (Y band blue-shifted, and the shoulder on the Soret band (-445 nm at neutral pH) disappeared while the small peaks at 550 and 520 nm became less pronounced.

Effects of Organic Cosoluents-Solutions of CcO(0)CO were dialyzed overnight against 0.1 M sodium phosphate buffer, pH 7.5, containing either 25% (v/v) ethylene glycol or 25% (v/v) glycerol. Neither polyhydric alcohol caused a significant change in the visible and Soret spectra. Furthermore, in the CO infrared spectra the uco and AUU values were unchanged in the presence of the cosolvents. Small changes in the relative intensities of the four CO conformers occurred on going from water to organic-water mixtures. The major band at 1963 cm" comprises 79% of the total area in H20 but 76% in 25% ethylene glycol or 25% glycerol. A corresponding increase is observed in the 1959 cm-' band representing 13% of the total area in H 2 0 but 17 and 16% in 25% ethylene glycol and 25% glycerol, respectively. There is also a significant increase in the area of the 1968 cm" band in 25% ethylene glycol (7% of total area), whereas no change is observed for this band in 25% glycerol (4.5% of total area). Varying the ionic strength of the medium from 10 mM to 1 M sodium phosphate had no observable effect on the infrared bands for CcO(0)CO.

Enzyme Actiuity-The enzyme activity of our preparations was examined as a function of temperature, pH, D20, ionic strength, and the presence of organic cosolvents. Temperature uersus activity measurements gave an enthalpy of activation value, E,, of 13 kcal/mol determined from Arrhenius plots (In activity uersus 1/T) (Fig. 9). The activity decreased logarithm- ically with increasing cosolvent concentration. The enthalpy of activation was not affected by ethylene glycol but was reduced slightly by glycerol (Table VI).

The activity (the first order rate constants) uersus pH profiles for three different enzyme preparations are plotted in Fig. 10. Duplicate activity assays were performed at each pH for all three preparations, with the second assay carried out 3-4 h after the first assay. The difference between the two assays reflects the decrease in activity observed with time at 24 "C. The solid lines represent an average of the two runs (Fig. 10). All three preparations show pH optima at 5.6-5.7 and 6.0. The maximal activity at both pH optima is 10.5 & 1 s-'/mg of protein/3 ml at 24 "C. A third pH maximum at 5.1- 5.2 was found for all three preparations. The oxidase reaction follows first order kinetics over the pH range studied. How- ever, from pH 4.5 to 5.4 lower correlation coefficients are observed (0.992-0.999) than between pH 5.5 and 7.5, where first order kinetics are strictly obeyed (correlation coefficients higher than 0.999). The lower correlation coefficients observed at the lower pH values reflect an apparent faster initial phase and a slower second phase. These observations may result from a rapid loss in activity at lower pH due to denaturation. The activity was also found to be highly dependent

13646 Mechanism of Dioxygen Reduction by Cytochrome c Oxidase TABLE I11

13C and isotope shifts in C - 0 stretch bands deconvoluted from infrared spectra of fully reduced cytochrome c oxidase carbonyls

Experimental conditions as in Table I except for use of only HzO as solvent. CI CII CIII CIV

Isotope Observed" Calculatedb Observed" Calculatedb Observed" Calculatedb Observed" Calculatedb

12C160 1955.2 cm" 1959.6 cm" 1963.5 cm" 1969.4 cm" 1911.8 1914.9 1916.1 1918.8 1919.9 1924.4 1925.7

1911.5 1908.1 1915.6 1912.4 1919.4 1916.2 1924.3 1921.9 1 3 ~ 1 6 0 1910.0 lZC180

As shown in Table I. *Calculated values as those expected from shifts from vco values observed for 12C160 based upon a simple

diatomic vibrator (13).

TABLE IV C-0 stretch band parameters from deconvolutwn of infrared spectra for carbonyls of cytochrome c oxidase at

different redox levels The spectra were obtained for solutions at 30°C and taken from Ref. 14.

Redox level" Band CI Band CII Band CIII Band CIV

yco Aulp Areab uco Aullr Areab uco Aulla Areab yco Aul12 Areab cm" i 0.4 cm" i 0.3 % i 0.5 cm" i 0.2 cm" i 0.2 % i 0.8 cm" i 0.2 cm" f 0.2 % i 0.8 cm" i 0.4 cm" f 0.3 % i 0.5

0 1955.2 4.0 2.7 1959.5 4.0 13.2 1963.3 4.2 79.9 1969.4 4.2 4.2 I 1955.5 4.2 3.0 1959.6 3.8 9.8 1963.6 4.6 82.3 1968.3 4.5 4.9 I1 1957 111

4.2 2.9 1961.2 4.5 18.5 1964.9 4.6 75.7 1970 4.2 2.9 1958.5 4.2 3.7 1962 4.2 16.7 1965.4 4.2 76.6 1969 4.2 3.0 Redox levels are designated 0, I, 11, and 111 for the species obtained when resting oxidase (as isolated) is reduced

with four, three, two and one electron equivalents, respectively. * Area represented and calculated as described in footnotes of Table I.

1 I I I I 1

I I I I 1 I 1980 I970 1960 1950

WAVENUMBER (cm")

FIG. 4. Effects of oxidation state on C-0 infrared stretch spectra of CcO carbonyls in 10 mM sodium phosphate buffer at pH 7.4 and 30 "C. The spectra from left to right represent the carbonyl complexes of 1-4 electron reduced species (14).

on ionic strength with an optimum range, 1 = 0.075-0.090, in sodium phosphate buffers at pH 6.0.

The activity is substantially lower in DzO reaction mixtures compared to HzO mixtures (Fig. 11). When CcO in either Hz0 or DzO buffers was injected into a DzO assay mixture, the activity was significantly lower than in HzO mixtures. The magnitude of the decrease in activity on going from Hz0 to DzO mixtures depends upon pH, with the greatest decrease occurring at the H20 pH optima, 50 and 66% at pH 5.65 and 6.0, respectively. Above pH 6.5 the activity was approximately equal in HzO and DzO. The activity versus pH profile in DzO has two pH optima which occur at lower pH (5.1 and 5.4) and exhibit much lower activity (5.2 and 6.4 s"/mg of protein/3 ml, respectively) than is observed in HzO. The differences in pH at which the two maxima appear in HzO and DzO may reflect the difference between pH and pD (pD = pH + 0.4) (28). However, since the activity at the optima in D20 was -40% less than at the corresponding optima, in HzO, the

I I I

I 1 I I I I980 1960 1940 1920

WAVENUMBER (cm") 1980 1960 1940 1920

FIG. 5. Effects of temperature on the carbonyl infrared spectrum of CcO(0)CO in HnO and DnO. Top, spectra of -2 mM CcO(0)CO in Hz0 (left) and D20 (right) 10 mM sodium phosphate buffers at pH 7.4 and 24 "C. Middle, spectra of CcO(0)CO in H10 (left) and DzO (right) a t 4 "C. Bottom, computer-generated difference spectra (low minus high temperature) in Hz0 (left) and DzO (right). The parameters of the theoretical bands used for deconvolution are listed in Tables I and 11.

observed DzO effect can not solely be attributed to the difference between pH and pD.

DISCUSSION

Observation of Multiple CO Stretch Bands in Cytochrome c Oxidase (0)CO"Two major bands, and also two minor bands

Mechanism of Dioxygen Reduction by Cytochrome c Oxidase 13647

A 43"CI 1 1 0.005 t - T

I I I I

1980 1960 1940 1920 WAVENUMBER (cm")

FIG. 6. Effects of elevated temperature on the carbonyl infrared spectrum of CcO(0)CO in 10 mM sodium phosphate DzO buffer at pH 7.4. top, spectrum after 21 h at 24 "C, 5 h at 4 " C , 15 h at 24 "C, and 50 h at 37 "C. Upper middle, the spectrum after heating the solution giving the top spectrum for 4 h at 43 "C. Lower middle, the spectrum after heating the solution giving the upper middle spectrum for an additional 18 h at 43 "C. Bottom, the spectrum after heating the solution giving the lower middle spectrum for 4 h at 52 ' C .

of uncertain significance, were found at all redox levels (Fig. 1, Tables I and IV). Expected isotope shifts were observed with lzC'sO and 13C'60 for each band (Fig. 3 and Table 111). As with hemoglobin and myoglobin carbonyls, the multiple CO infrared bands of oxidase carbonyls appear to be due to

discrete protein conformer structures at the oxygen reaction site. Relative integrated intensities of the bands reflect the relative stabilities of the conformers. Infrared spectra for CO bound to mitochondria from various sources also reveal the presence of two bands at -1963 and -1959 cm", with an indication of smaller bands at higher and lower frequencies less certain in view of the low S/N (29).

CO Stretch Band Widths-Aulh values vary from 20 to 30 cm" for protein-free heme carbonyls, from 7 to 10 cm" for hemoglobin carbonyls, and from 8 to 18 cm" for myoglobin carbonyls (Fig. 2 and Table 11). A bandwidth of -25 cm" has been observed for a cytochrome P-450 carbonyl (30). The CO vibrators for a given protein conformer exhibit a distribution of stretch frequencies because the CO molecules are in slightly different environments. The width of the band is then a measure of the diversity of these environments, the range of microstructures in the protein near the CO ligand. The correlation between bandwidth and the degree of environmental diversity has been clearly demonstrated by the infrared spectra of nitrous oxide in a variety of solvents; the width of the antisymmetric stretch band for N20 increases with an increase in the conformational flexibility of the solvent molecule (31). The small bandwidth in CcO carbonyls compared to other hemeproteins is clearly due to smaller inhomogeneous broadening in CcO as a result of less diversity in the environment (protein residues or intraprotein solvent) at the 0 2 -

binding site. Therefore, a conformationally stable, highly organized "oxygen pocket," well isolated from the external medium is present in CcO.

Effects of Overall Redox Level on CO Stretch Bands-Fre- quencies, bandwidths, and relative stabilities of the C-0 stretch bands exhibit small but measureable differences among the four redox states (Fig. 4 and Table IV). The changes in frequency due to changes in oxidation state of redox active metals other than FeL are surprisingly small in view of the widely observed high sensitivity of the carbonyl band frequency of metal carbonyls in either solvent or ligand structure. Solvent polarity markedly affects vco of protein- free heme carbonyls; e.g. N-methylimidazole protoheme di- methyl ester carbonyl gives vco values of 1980,1969, and 1959 cm" in CCL, benzene, and CH2ClCH2C1, respectively (13). The effects of dipolar character of solvating molecules on stretch frequencies are clearly shown by the solvent effects on N20 infrared spectra referred to above (31). If the assumed interatomic dis:ance between iron and copper at the oxygen pocket of 3-5 A is correct (32, 33), changes in the oxidation state of the Cu might well cause large shifts in the vco for CO bound to the adjacent heme iron. The observation of only small shifts suggests either that compensating medium effects insulate CO from the effects of change in oxidation state at copper, or that the copper and iron are not as close in the reduced forms of the enzyme as the resting oxidase extended x-ray absorption fine structure results suggest (32, 33).

Effects of D20/H20 Exchange on CO Stretch Bands-The infrared spectra of CcO(0)CO obtained in D20 over a range of temperatures exhibit four CO stretch bands with shifts of approximately -0.2 cm-' compared to the spectra obtained in Hz0 (Table I). With HbACO substitution of DzO for H20 affects neither the frequency nor the shape of the CO infrared bands at various pH values (17). The exchange of HzO by D20 has little effect on the 0-0 infrared spectrum in oxy- myoglobin where H-bonding to the O2 ligand is suggested by several lines of evidence (34).

Effects of Temperature on CO Infrared, Visible, and Soret Spectra-There are small, but detectable, changes in the relative intensities of the two major CO infrared bands at 24


FIG. 7. Effects of temperature on visible and Soret spectra of CcO(0)CO in 10 mM sodium phosphate buffers, pH 7.4. Spectra were recorded at the temperatures shown. The top spectrum at 24 "C represents CcO(0)CO at the start of the experiment. The second spectrum of CcO(0)CO at 24 "C (third spectrum from the top on the left) is recorded after 6 h at 4 "C (second spectrum from the top) and an additional 20 h a t 24 "C. The 37 "C I and 37 "C I1 spectra are recorded after 5 and 28 h of CcO(0)CO at 37 "C, respectively. The 43 and 52 "C spectra were recorded after 16 and 5 h at the two temperatures, respectively.

w 0 z a m CK 0 m m a 7 0.057

0 . 0 5 I

37°C II

r I I I I I I

400 500 600 700 400 5 0 0 600 WAVELENGTH i n r n ) WAVELENGTH (nm)

uersus 4 "C (Fig. 5). The reversibility of the temperature (Table 11). Decreasing the temperature from 30 to 4 "C of changes between 24 and 4 "C (total integrated area remains either crystals or solutions of HbA and Hb Zurich carbonyls constant) indicates that the two major conformers are inter- shifts intensity from minor bands to the major bands (19). convertible and are not due to heterogeneous or denatured Similar temperature effects have been observed for other protein preparations. Similar but greater temperature effects hemoglobin and myoglobin carbonyls (Table 11) (18, 25). have been observed for hemoglobin and myoglobin carbonyls The changes observed in the CO infrared spectra of


TABLE V C - 0 stretch band parameters for the carbonyl of fully reduced

cytochrome c oxidase at different pH values Data are based on the deconvolution of spectra of Fig. 8.

pH Band CII Band CHI

yco AullP Area" uc0 Area" cm" k 0.3 cm" k 0.5 ?& cm"f 0.3 cm" & 0.5 %

10.8 1959.4 4.5 20 1963.4 5.0 80 10.3 1959.6 4.5 19 1963.0 4.5 81 9.8 1959.5 4.0 12 1963.1 4.3 88 8.8 1959.2 4.0 13 1963.1 4.5 87 7.4 1959.3 4.0 14 1963.3 4.2 86 6.0 1959.3 4.0 7 1963.2 4.6 93 5.4 1959.3 4.0 7 1963.2 4.0 93 Band area as a percentage of the sum of areas of bands CII and

CIII. Due to low S/N ratio and the consequent difficulty in base line slope correction these values may be subject to as much as 10% error.

CcO(0)CO at temperatures of 37 "C and above indicate that an irreversible denaturation of the enzyme has occurred (Fig. 6). The increase in intensity of the minor bands at 1955 and 1969 cm" at 37 "C raises the question whether these bands represent partially denatured enzyme. However, the small bandwidth of -4 cm" of these bands is much less than is generally observed for denatured protein products ( AuH - 20 cm") (13). Therefore, it is concluded that the minor bands represent conformers of native CcO. On the other hand, the appearance of a new band at -1973 cm" probably does represent denatured enzyme. Incubation at 37 "C intensifies and broadens this new band. The infrared, visible, and Soret spectral changes observed for c c o a t 37 "c occur slowly (2-3 days) (Figs. 6 and 7). Thus, the denaturation process under these conditions is gradual rather than abrupt. From 43 to 52 "C the appearance of a broad band centered at 1970-1975 cm" with AvH - 20 cm" indicates substantial, irreversible denaturation (Fig. 6).

Effects of pH on CO Infrared, Visible, and Soret Spectra- The sensitivity of CO infrared, visible, and Soret spectra to changes in pH differs considerably among CcO, Hb, and Mb carbonyls (Table 11). Only minor changes are observed in the pH range 4.5-10.8 for the two major CO infrared bands of CcO(0)CO (Fig. 8). Larger shifts are observed in the relative intensities of the CO stretch bands in hemoglobin and myoglobin carbonyls over a similar pH range (Table 11) (17, 24, 25). The vco and AvH values are unchanged over the pH ranges studied for all three proteins (Table 11), suggesting that relative stabilities, but not the structures of the individual conformers, change with pH. Similarly, the visible and Soret spectra of CcO(0)CO remain unchanged from pH 5.4 to pH 10.3, whereas significant changes occur at pH extremes.

Effects of Temperature on Activity-The dependence of oxidase activity on temperature, we found, is in general agreement with reports on mitochondrial membranes, CcO reconstituted into vesicles, and detergent-solubilized enzyme (35- 42). However, we did not observe the reported break in the Arrhenius plot between 20 and 25 "C attributed to temperature-induced conformational changes (38, 40). Neither our data (Fig. 9) nor the data of Robinson et al. (42) require an interpretation with two lines intersecting between 20 and 25 "C. Furthermore, the insensitivity of the CO infrared spectra to changes in temperature between 4 and 24 "C (Fig. 5) indicates that no major temperature-induced conformational changes occur near the ligand-binding site.

Effects of pH and Ionic Strength on Activity-Previous reported pH optima vary from pH 5.7 to 7 (43-49). Our observation of two pH optima (at pH 5.65 and 6.0) has not been reported previously (Fig. 10). Only a single pH optimum

in this range has been reported (43-45). We conclude that the two pH optima found with three different enzyme preparations represent an intrinsic property of the enzyme and reflect a complex pH dependence involving several proton donating groups with pK, values between 5.0 and 6.5 (Fig. 10). The closeness of the two pH optima suggests cooperativity among the different proton donors. The pH profile also suggests that pH affects kcat, rather than K,,,. A broader pH optimum with pK, values close to pH 4 and 9 would be expected if the negatively charged carboxyl and positively charged amino groups which are involved in the electrostatic interactions between cytochrome c and CcO determined the rate of the reaction. Earlier observations (50,51) that pH strongly affects kat, but not kat/K,,,, also support a pH effect on the catalytic steps and not on the rate of combination of cytochrome c with CcO as reflected in the K,,, values. Two pH optima suggest that different steps involving protonation at intermediate stages during the catalytic cycle become rate limiting at different pH values. The rate constants for electron transfer in these steps could also be pH dependent because of protonation effects remote from the 02-binding site, a possibility supported by the lack of pH effects on CO infrared spectra of CcO(0)CO over the pH range 4.5 to 10.8.

Effects of D20/H20 Exchange on Activity-Substitution of H20 by D20 decreases the steady-state activity of CcO by 40- 60% for mitochondria (52-55) as well as reconstituted (51) and solubilized enzyme preparations (40, 56). D20 reportedly decreases kat by 50%, but has no significant effect on kat/Km (51), indicating that D20 may affect the rate of the catalytic steps in the dioxygen reduction rather than the interactions between cytochrome c and CcO. Our D20 exchange studies support the involvement of protons in the rate-limiting step(s) in O2 reduction and, in agreement with earlier workers (56), show a gradual decrease in the catalytic activity of CcO at pH 6.0 as the D20 content is increased.

Effects of Ethylene Glycol and Glycerol on Actiuity-The observed decrease in activity with increasing organic solvent concentration (Table VI) is consistent with earlier reports of solvent inhibition of reactions of hemeproteins which involve proton uptake (53, 56-58). The activation enthalpy is nearly the same in H20, 25% ethylene glycol, or 25% glycerol (Table VI) implying that the rate-limiting step of the electron transfer is independent of the nature of the surrounding solvent. However, the maximum rate differs significantly with medium and, therefore, the solvent environment may affect the en- tropy of the rate-limiting step, e.g. by the ordering of solvent molecules. The organic solvents do not affect the CO infrared spectra which argues against solvent-induced conformational changes at the CO-binding site. The depression of oxidase activity by glycerol and ethylene glycol may explain their inhibition of mitochondrial respiratory chain activity (53, 56, 58).

Significance of the Lack of Correlation of pH and D20 Effects on CO Infrared Spectra and Actiuity-No obvious correlation has been detected between the CO infrared data and enzy- matic activity. Sharp activity maxima are centered at pH 5.6- 5.7 and 6.0, but there is no frequency shift or broadening of the two major CO stretch bands over the pH range 4.5 to 10.8. Ionizable groups close to the CO (02) binding site appear either absent or totally isolated from solvent. Retention of the CO stretch band shape over a broad pH range indicates no large pH-induced stereochemical changes in the local binding environment. The broadening of the infrared spectrum at high pH (>lo) may result from exposure of the CO ligand to solvent water.

Nature and Physiological Significance of Conformer Struc-


FIG. 8. Effects of pH on carbonyl infrared spectra of CcO(0)CO in 10 mM sodium phosphate buffer at 24 OC. The reference cell contained the same buffer as the sample cell. Each spectrum is an average of 3-4 single scans and, except for pH 4.4 and 11.4, are slope corrected. Parameters of theoretical bands used for deconvolution are listed in Table V.

I I I I I I I

1980 1960 1940 1980 1960 I940

WAVENUMBER (crn")

tures-The detailed structures of the individual conformers are not known but some features can be inferred. The frequency and the intensity of the four CO bands in CcO can be influenced by (a) the identity and orientation of the ligand trans to CO, ( b ) the electronic properties (basicity) of the porphyrin, (c) steric interactions imposed by the immediate environment about the CO ligand, and ( d ) the dipolar and dielectric nature of the CO environment (12,13). The bonding of CO to heme a3 mimics that of O2 but CO prefers linear Fe- C-0 bonding with CO normal to the heme plane whereas 0 2

prefers a bent-end-on stereochemistry (34,59). The O2 ligand is expected to experience the same inflexible, uniform, and isolated environment that is indicated for CO by the CO infrared spectra. However, since 02, not CO, is the substrate, the CO ligand can be expected to be forced by the protein pocket to deviate from the normal to the heme plane geometry, analogous to what is found in crystals of myoglobin and hemoglobin carbonyls (60,61). The tight and inflexible nature of the oxidase ligand-binding site can result in steric con-

straints from nearby amino acid residues to cause tilting and/ or bending of the Fe-CO bond. A Raman spectroscopic study of CcO(0)CO suggested an FeCO angle of 175" k 5" with the CO tilted from the normal to the heme plane (62). However, the Raman study did not detect multiple conformers.

The dissimilar CO spectral behavior observed for HbCO, MbCO, and CcO-CO at various pH values and temperatures (Fig. 2 and Table 11) no doubt reflects the different structural requirements of these proteins for carrying our their physiological functions. Hb and Mb need rapid access of 0 2 to and from heme iron for efficient delivery of O2 from the lungs to cytochrome c oxidase in tissue. A flexible, dynamic protein structure at the 02-binding site is required to permit O2 to enter or leave without reduction of bound 02. CcO, on the other hand, requires a ligand-binding site structure that promotes the reduction of O2 to water without production of partially reduced species (HzO2 or oxyradicals) as unwanted side products. The maintenance of a conformationally stable geometry at the dioxygen reduction site throughout the elec-


1.0 I I 3, 3.2 3.4 3.6

T"x IO ' (OK") FIG. 9. Arrhenius plot of enzyme activity versus tempera-

ture.

TABLE VI Influence of organic cosolvent on the activation energy and the

oxidase activity at different temmratures

Organic cosolvent (%, v/v) % Control activity" Activation energyb

37°C 26'C 19°C E.4

keal/mol Glycerol 5 85 78 106 11.5

15 55 59 73 10.7 25 43 43 47 12.6

11.6 f lnaverage)

Ethylene glycol 5 60 57 62 13.1 15 26 28 23 14.4 25 10 12 10 13.1

13.5 & Owaverage) Control in 0.1 M phosphate, pH 6.0, at specified temperatures. From In activity versus 1/T plots; controls (i.e. without added

cosolvent) gave E,,$ = 13.4 kcal/mol.

tron and hydrogen transfer steps of the dioxygen reduction reaction can be critically important to rate enhancement by the enzyme.

Dioxygen Pocket Structure and Enzyme Catalysis-In the oxidase reaction four electrons and four hydrogens are do- nated to O2 to produce two HzO molecules (reaction l). Each of several processes, oxygen binding, electron and hydrogen transfers to oxygen, cleavage of the 0-0 bond, and release of two water molecules, must be carried out rapidly (63). CcO promotes these various steps utilizing an active site structure which permits facile conversion of dioxygen to water, via steps which must follow an efficient low energy pathway. The results reported above combined with consideration of the multiple electron and hydrogen transfers required to carry out reaction 1 and present views of CcO structure, support the catalytic mechanism for O2 reduction shown in Scheme 1. O2 is expected to bind in a bent-end-on stereochemistry (34) to five coordinated Fez+ (Structure 11). Designating histidine as proximal ligand stems from an 16N histidine EPR study of the yeast oxidase in the fully oxidized state which shows that an endogenous imidazole group ligates heme u3 iron (64).

A stable oxygen-binding site with limited conformational flexibility and well secluded from the aqueous environment suggests that protein residues immediately adjacent to the

I I I I I I I I

12 -

IO -

S -

6-

4 -

2 -

12 c

I2i 0

1 I I I I I

5.0 6.0 7.0 8.0 PH

FIG. 10. Dependence of oxidase activity on pH. The activity assays were carried out at 24 "C in 0.1 M sodium phosphate buffer. The three panels represent data for three separate enzyme preparations. Open and closed circles represent the first and second runs of the duplicate assay, respectively. A solid line connects the average values for the two runs.

bound O2 serve as H-donors. The closeness of CUL to FeL can place ligands bound to the CUL at a suitable distance and in the correct orientation to facilitate electron and hydrogen transfers to the FeL-bound 02. For these reasons the nearest neighbor atoms to the O2 ligand in the oxy Structure I1 are hydrogens. These hydrogens should be located at sites suitable for facile covalent bonding to oxygen atoms as the reduction of O2 proceeds. Hydrogen bonding between XH, YH, and ZH and the bound dioxygen will stabilize the ligand site structure and restrict the rotation of 0 2 about the Fe-0 bond. The identities of X, Y, and Z are unknown. The restraints upon the translational, vibrational, and rotational degrees of freedom of O2 by tight binding within the pocket will contribute substantially to rate enhancement of reaction 1. (The restric- tion of these degrees of freedom is much more difficult to


5.0 6.0 7.0 pH

FIG. 11. Dependence of oxidase activity on pH in Ha0 and DzO 0.1 M sodium phosphate buffers at 24 OC. The plots represent the average values for two runs in HzO (circles) and in DzO (squares).

achieve with a small diatomic substrate such as O2 than with a large substrate.) Hydrogen bonding between the 0 2 ligand and RIXH, &YH, and R3ZH of Structure I1 will not only stabilize the oxygen pocket structure but will also close the pocket to access from the external medium.

Structure IV can result from the transfer of a total of three electrons to Structure 11. One electron from oxidation of iron (11) to iron (111) plus three additional electrons provide the four electrons needed to reduce both oxygen atoms of 0 2 to the redox level of water. Structures 11-IV illustrate a way in which the complete reduction of 0 2 (Steps 2 and 3) can be accompanied by only very small shifts in the locations of the reacting atoms within the oxygen pocket. Although Structure IV is designated open in Scheme 1 because the 0-0 bond has been broken and a water molecule may be released from the pocket, the structure of the pocket as shown remains in the closed position. Loss of Hz0 from Structure IV to the outside gives Structure V which opens up the pocket to external reactants.

The steps for electron and hydrogen transfers between the oxy species (Structure 11) and the first species in which the oxygen atoms are fully reduced (Structure IV) of Scheme 1 may involve a hydroperoxy intermediate (Structure 111). Structures 11-IV indicate a role for the copper associated with heme a3 and also suggest a role for zinc although there is no direct evidence that Zn2+ is in fact located at the O2 reduction site (8,9). It is assumed that an electron pathway is available for electron transfer from cytochrome c to both FeL and CUL. The Fe to Cu distance is considered to remain nearly constant during Steps 2 and 3. All components in the oxygen pocket are assumed to remain in similar positions with respect to each other, an assumption supported by the insensitivity of the CO stretch band to changes in redox level (14).

The reduction of Fe2+02 to form Fe3+00H in Step 2 can be accomplished with the addition of one electron to Cu2+ and subsequent transfer of an electron from Cu+ to the 02 ligand via the H-bonded network (&YH) and a concerted inner sphere one electron transfer from Fez+ to 0 2 , in analogy to the reaction of oxyHb with Fe2+(CN)6Hz03- (15, 65). The electron flow may be formally considered to proceed from Cu+ via Y to H to the terminal oxygen of 02. The overall shifts in location for the atoms involved (Fe-0-0-H-Y-Cu) during Step 2 can be very small. Holding the reacting atoms for a sufficient length of time in the precise stereochemical orientations and at the interatomic distances needed to permit vibrational activation is critically important for rate enhancement by an

enzyme (66). The hydrogen and electron transfers in Step 2 from Y to the terminal 0 of 0 2 may be described as either a hydrogen atom transfer (H . ) or as the transfer of H+ plus e-. Structure I11 is closed to access by reactants from the external medium and can explain why H202 is not observed to leak from the oxidase under turnover conditions.

The covalent bonding of the hydrogens of H-X and H-Z to the oxygen atoms of the hydroperoxide is shown to accompany 0-0 bond cleavage (Step 3). The 0. . . . H-X and 0 . . H- Z hydrogen bonding promotes 0-0 bond cleavage (Structure 111). If present, zinc as a dipositive ion could render the hydrogen on Z acidic and the donation of a proton by Z will promote 0-0 bond cleavage.

By maintaining a nearly constant interatomic distance between Fe and Cu throughout the catalytic cycle, i.e. from Structure 11-IV in Scheme 1, the rates of electron and hydrogen transfers are increased by reducing the entropic contri- bution to the activation energy due to conformational changes and by providing precise stereochemical control at the active site. Cleaving the 0-0 bond increases the distance between oxygen atoms in Structure IV to permit loss of one Hz0 molecule to the external medium. Steps 4-6 of Scheme 1 represent a reasonable route from IV to I. Structure I, like deoxyHbs and deoxyMbs, is a five-coordinate iron (11) species which reacts with O2 to give 11. The oxygen pocket must be open in I to allow the O2 molecule entry from outside the protein. To go from IV to I, four protons and one electron must be added; two water molecules must leave. The exact sequence from IV to I cannot be defined with certainty but the sequence shown is chemically reasonable based on known hemeprotein bioinorganic chemistry. In Steps 5 and 6 the release of the second H20 molecule from an open pocket occurs only after the iron is reduced, in analogy to the loss of water upon reduction of aquometMb to form five coordinate deoxyMb. Opening the active site to the external aqueous medium at the end of the catalytic cycle allows reprotonation of the three hydrogen donors, R,X, &Y, and R3Z. In this way, Structure I is regenerated from IV and is poised to receive an O2 ligand with three donatable hydrogens to enter a new catalytic cycle.

Concluswn-The structure of the dioxygen reaction site and the factors that alter enzyme activity in cytochrome c oxidase purified from bovine heart have been investigated. Visible, Soret, and carbonyl infrared spectra reveal a ligand- binding site at heme u3 iron that is extraordinarily stable and well isolated from the external medium when CO is bound. The effects of DzO and pH on oxidase activity support rate- limiting hydrogen transfers during the reduction of 0 2 to water. A novel mechanism consistent with present evidence involves bent-end-on bonding of O2 to Fez+ within an unusually immobile environment with potential hydrogen donors immediately adjacent to the 0 2 ligand. An appropriate stereochemical arrangement of H-donors, a redox-active Cu, and possibly Zn2+, facilitates the rates of electron and hydrogen transfers required to complete the oxygen atom reductions, 0-0 bond cleavage, and water formation steps within an isolated protein pocket. The stability and isolation of the 0 2

reaction pocket can provide the required rate enhancement and prevent partially reduced 0 2 products, i.e. superoxide, hydrogen peroxide, and hydroxyl radical, from leaving the site.

Acknowledgments-We thank Professor Shmya Yoshikawa, Dr. William H. Woodruff, and Dr. Sharon Sowa for helpful discussions, Dr. Jo Hazzard for assistance with hemoglobin and myoglobin carbonyl infrared Soret spectra, and Diana Davis for preparation of the manuscript.

SCHEME 1


(open) H\z/R3 V I

?

/ \

(open) *\/E3 V

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