Infrared Evidence of Azide Binding to Iron, Copper, and Non-metal

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 14, Issue of May 15, pp. 9757-9766,1992 Printed in U. S. A .

Infrared Evidence of Azide Binding to Iron, Copper, and Non-metal Sites in Heart Cytochrome c Oxidase*

(Received for publication, August 14, 1991)

Shinya YoshikawaS and Winslow S. Caugheys From the Department of Biochemistry, Colorado State University, Fort Collins, Colorado 80523

Interactions of azide ion with bovine heart cytochrome c oxidase (CcO) at five redox levels (IV) to (0), obtained by zero to four electron reduction of fully oxidized enzyme CcO(IV), were monitored by infrared and visible/Soret spectra. Partially reduced CcO gave three azide asymmetric stretch bands at 2040, 2016, and 2004 cm” for CcO(III)N3 and two at 2040 and 2016 cm” for both CcO(II)N3 and CcO(I)N3. Resting CcO(1V) reacts with Ng to give one band at 2041 cm-l assigned to Cug+N3 and another at 2051 cm” to Ng that is associated with protein but is not bound to a metal ion. At high azide concentrations the weak association of many azide molecules with non-metal protein sites was observed at all redox levels. These findings provide direct evidence for 1) N, binding to CUB as well as Fe., in partially reduced enzyme, but no binding to Fen, in fully oxidized enzyme and no binding to either metal in fully reduced enzyme; 2) a long range effect of the oxidation state of Fen or CUA on ligand binding at heme a3, but not at CuB; and 3) an insensitivity of either Fees or CuB ligand site to changes in ligand or oxidation state at the other site. The observed independence of the Feas and CUB sites provides further support for Fez: OOH, rather than Fez; OOCug+, as an intermediate in the reduction of 0 2 to water by the oxidase.

Azide has been known to be a potent inhibitor of cytochrome c oxidase (CcO)’ since the pioneering studies on respiration by Keilin and Hartree over 50 years ago (1). This inhibition, as with the respiratory inhibitors carbon monoxide and cyanide, is considered to result from azide binding to the iron of heme a3 at the dioxygen reduction site in the oxidase. However, distinctly different effects of the binding of azide compared with CO and CN- have been noted (2). For example, azide has little or no effect on the visible/Soret spectra of either fully oxidized or fully reduced enzyme in contrast to the marked effects of CO and/or CN- (3-6). On the other

*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.

$Permanent address: Dept. of Life Science, Himeji Institute of Technology, 1479 Kamigohri, Akoh, Hyogo, 678-12, Japan.

J To whom correspondence should be addressed. ’ The abbreviations and trivial names used are: CcO, cytochrome c

oxidase; CcO(IV), CcO(III), CcO(II), CcO(I), and CcO(0) represent cytochrome c oxidase at different oxidation states, the fully oxidized, one-, two-, three-, and four-electron reduced species, respectively; Fe.,, the iron that binds external ligands, CUB, the copper most closely associated with Fe,; Fe, and CuA, the iron and copper involved in the electron transfer from cytochrome c to the O2 reduction site, Cux, copper in excess of iron that is intrinsic and not adventitious.

hand azide does markedly alter the visible/Soret spectra when the oxidase is partially reduced as do CO and CN- (7, 8). At present, the sites of azide binding and the effects of redox level on binding are poorly understood.

The enzyme purified from bovine heart is now recognized as having several potential metal sites for ligand binding, two heme irons (Fe, and Fea,), three coppers (CUA, CUB, and CUX), zinc, and magnesium (9-11). Recent infrared studies of CO and CN- binding to CcO at the five different redox levels that result as a consequence of four redox active metal centers (Fe,, Fe.,, CuA, and CuB) provide direct evidence of the metal to which CO and/or CN- bind and of the oxidation state of the metal ion involved. For example, measurements of the CO infrared stretch band show stable CO binding only to one reduced heme (Fe::) at all partially and fully reduced redox levels and no CO binding to the fully oxidized enzyme CcO(1V) (12-14). In contrast, CN- infrared spectra show CN- binding to Fe,, and/or CuB with the oxidation state of the metal ion as well as the metal selected, dependent on the overall redox level of the enzyme (15, 16). Changes in the oxidation state or the ligand at either Fe,, or CUB have little effect on the other ligand site at redox levels other than CcO(IV), even though the two metals are very near each other, less than 5 A apart (17, 18). The demonstration of CN- binding to CUB as well as Fee, and the lack of interaction between the Fee, and CUB ligand sites are highly relevant to the likely participation of the two metals in dioxygen reduction and provided support for new concepts of the mechanism of enzyme catalysis (14, 16, 19). Direct infrared evidence of the binding of azide, an anion which compared to cyanide is larger and less nucleophilic (20) and prefers bent-end-on to linear bonding at metal ions (21), can be expected to provide further insight into the structure and function of the dioxygen reduction site.

The few available reports on vibrational spectra of azide bound to metal sites in proteins indicate these spectra with bands near 2000-2100 cm” can provide important information on metal site structures and properties (22-26). These studies include monitoring of spin-state equilibria in methe- moglobin and metmyoglobin azides (22, 23, 25) and explora- tions of the copper sites in hemocyanin (26). Infrared studies of non-protein azides provide useful reference spectra; e.g. azide band parameters are shown to be sensitive to porphyrin and trans ligand structures and to solvent in hemin azides (25, 27, 28) and to other ligands in copper azide complexes

Here we report an infrared study of azide binding to bovine heart CcO at five different redox levels. Binding of azide may occur at both Fe., and Cug but not to the reduced metal ions. This binding to metals is highly dependent on the overall redox level of the enzyme: at Cui+, but not FeX:, when fully oxidized, at both Cui+ and Fez: when partially reduced, and no binding when fully reduced. Azide bonding to Fez: is affected by the oxidation state of Fe, to CuA. Association of

(21, 29-31).

9757

9758 Azide Binding to Iron and Copper Sites in Cytochrome c Oxidase

azide with protein at non-metal sites is detected at all redox levels.

MATERIALS AND METHODS

Cytochrome c oxidase was prepared from fresh bovine heart as described earlier (12). Isotopically labeled potassium azide (K1'N14N14N) was a product of ProChem. Visible/Soret and infrared spectra were measured with Cary 17 and Perkin-Elmer 180 spectro- photometers, respectively. Infrared spectra were obtained for solutions at 17 "C in cells with CaF2 windows and a path length of 0.052 mm. The difference in path length between sample and reference cells was less than 2%. Resolution was maintained at 3 cm" or greater to enhance the signal-to-noise ratio. The buffer contained 10 mM sodium phosphate, pH 7.4, unless stated otherwise. Other experimental conditions are as described earlier (16). The concentration of CcO is expressed in terms of heme A as determined using c at 605 nm of 19.9 mM" cm" for the fully reduced enzyme (12). Infrared spectra are averages of at least 16 accumulated single scans.

RESULTS

CcO(1V) Azide-Upon the addition of 2 pl of 1 M sodium azide to 2.0 ml of 9.7 p~ CcO(1V) in 50 mM sodium phosphate, pH 7.4, only small changes in electronic spectra were observed, a slight intensification of the a band and a small red shift in the Soret band (Fig. 1, Table I). These spectral changes occurred within several minutes after the addition of azide. The minimal concentration of azide required to achieve the maximal spectral change was -100 p ~ . Similar spectral changes were observed for highly concentrated (-2 mM) CcO(1V) in 10 mM sodium phosphate, pH 7.4, in an infrared cell with CaFz windows. The final spectrum is similar to the one reported by Wever et al. (3), although the changes in spectra are in the opposite direction due to differences in the spectra prior to the addition of azide. Variations in the visible/

I I I I I I 1

I I I I 1 I I I 1 375 400 425 450 470 500 550 600 650 700

WAVELENGTH (nrn)

FIG. 1. Effects of azide on the visible/Soret spectrum of CcO(1V). A solution of the fully oxidized resting enzyme at 9.7 PM in heme in 50 mM sodium phosphate buffer, pH 7.4, in a cuvette (1- cm pathlength) was treated with 1 mM sodium azide at 23 "C. The dashed line shows the spectrum of the enzyme treated with azide and the solid line the spectrum of the enzyme prior to exposure to azide.

Soret spectra of different preparations of resting oxidized CcO are well documented (32-34). Similarly, the changes in spectra we observed upon exposure of CcO(1V) to azide varied among different preparations and with the freshness of the preparation. With some preparations no change in visible/Soret spectrum was found even at azide concentrations as high as 10 mM. The changes shown in Fig. 1 are as large as we have found for a fresh enzyme preparation.

The infrared spectrum of a solution of 2.2 mM CcO(1V) and 3.6 mM azide exhibited two bands at 2051 and 2041 cm" (Fig. 2, Table 11). The spectrum of Fig. 2 was obtained with dithi- onite reduced enzyme [CcO(O)] and azide in the reference cell at the same protein and ligand concentrations as in the sample cell. As shown below, no significant binding of azide to metals in CcO(0) was observed. Much of the base-line curvature of Fig. 2 is expected to result from reduction of the concentration of free azide due to azide binding to metal sites in the protein in the sample cell; the free azide band at 2047 cm", AvH = 21.3 cm", with E = 1.28 mM" cm" is much wider and more intense than the bound azide bands. Interpretation of these spectra is further complicated in that the amounts of free azide ion in the sample and reference solutions are not simply the difference between added azide and metal-bound azide, since, as discussed below, some azide associates weakly with protein at non-metal sites (Table 11). The metal binding sites appeared saturated with azide at concentrations of azide greater than 3.1 mM. The infrared band parameters of the 2051 and 2041 cm" bands for CcO(IV)N3 were consistent for different enzyme preparations, in contrast to the differences found in visible/Soret spectra. Thus, the small changes in visible/Soret spectra shown in Fig. 1 are unlikely to be associated with the azide binding that causes the 2051 and 2041 cm" bands of Fig. 2.

Partially Reduced CcO Azides-The effects of azide on the visible/Soret spectrum of partially reduced species of CcO were also examined (Fig. 3, Table I). CcO(III), CcO(II), and CcO(I), the 1-, 2-, and 3-electron reduction products from the fully oxidized species CcO(IV), were prepared as reported earlier (13, 16). The visible/Soret spectrum at 7 min after addition of 3.4 mM azide to 2.0 mM CcO(II1) exhibited an a- band at 603 nm and a Soret band maximum at 425 nm (dashed line in Fig. 3A). The spectrum changed with time; after 60 min a stable spectrum with bands at 601 and 429 nm was observed. No changes in spectrum beyond 7 min following addition of azide to CcO(I1) and CcO(1) were observed. The CcO(I1) azide exhibited an intense a-band at 602 nm and a Soret band at 440 nm with a shoulder near 430 nm (Fig. 3B); this spectrum is similar to the aerobic steady state spectrum of the enzyme when inhibited by azide reported by Gilmour et al. (8). The visible spectrum of CcO(I)N3 (Fig. 3C) is essentially the same as that of CcO(II)N3, whereas the Soret band maximum for CcO(I)N3 is substantially more intense

TABLE I VisiblelSoret spectral data for cytochrome c oxidase at five redox levels in the presence of azide

Experimental conditions are those of Figs. 1 and 3. Redox level X,. (relative intensity)

nm

IV 650" (0.027), 599 (0.108), 548 (0.099), 419 (1.00) 111 660" (0.014), 601 (0.191), 550 (0.112), 515 (0.112), 428 (1.000) I1 602 (0.311), 533 (0.126), 515 (0.133), 440 (1.000), 430" (0.868) I 602 (0.233), 565 (0.090), 515 (0.073), 442 (1.00), 420" (0.435) Ob 604 (0.195), 568" (0.077), 519 (0.066), 444 (LOO), 420" (0.364)

Shoulder. * Spectrum for CcO (0) in the presence of azide is not significantly different from the spectrum in the absence of azide.

Azide Binding to Iron and Copper Sites in Cytochrome c Oxidase

1 I 2080 2040

WAVENUMBER (cm") 2000

FIG. 2. Infrared spectrum of CcO(1V) azide. The fully oxidized resting enzyme, 2.2 mM in heme in sodium phosphate buffer, pH 7.4, was treated with 3.6 mM sodium azide. A mixture of 2.2 mM Cco(0) and 3.6 mM sodium azide in the same buffer was in the reference infrared cell.

TABLE I1 Infrared parameters for azide bands of azido cytochrome c oxidase at

four redox levels The infrared parameters for each band were determined from the

deconvoluted bands except for the 2042 cm" band at 25 mM azide and the free azide ion.

Redox level

ern" mM" crn" IV 2051 7.8 0.22

2041 5.2 0.26 111 2047 18 0.50'

2039 10 0.30 2027 6 0.08' 2016 13 0.33 2004 11 0.38

2040 8 0.23 2016 13 0.63

I 2047 22 0.40' 2040 8 0.15 2016 13 0.46

IV to I at 25 mM azide 2042 22 14.3 Free azide ion 2047 21.3 1.28

I1 2047 22 0.96'

' L values calculated on the basis of the bound azide concentration being equal to one-half the heme A concentration except for the free azide ion where the actual azide concentration was used. ' These values are those obtained in Figs. 5-7. The extinction of

the 2047 cm" band at any partially reduced redox level was between 0.40 and 1.0 mM" cm" depending on the preparation in the reference cell (see text).

e The 2027 cm" band intensity of CcO (111) shown is the weakest case. The strongest band intensity was 0.32.

with a less intense shoulder near 420 nm. The spectra for CcO(I)N3 and unliganded CcO(O), the fully reduced enzyme, are similar but detectably different (Fig. 3). No change in CcO(0) spectrum was observed upon addition of azide up to 25 mM. The differences in absorbance for 602 minus 650 nm and 442 minus 480 nm normalized to 2.0 mM enzyme at different redox levels are shown in Fig. 4. The absorbance difference values for levels (IV) and (0) are essentially those for CcO(1V) and CcO(0) free from azide. With increasing reduction, the a-band absorbance difference reached a maximum at level (11), whereas the Soret band absorbance difference increase was approximately linear from (IV) to (0). A small decrease in the absorbance difference for 602 minus 650 nm due to a red shift of the a-band was clearly observed upon reduction of CcO(I)N3 t o CcO(0). The effects of redox level on visible/Soret spectra shown in Fig. 4 have not been reported previously.

The infrared spectra of partially reduced azide species exhibited multiple azide bands. After the slow changes in the

1 ' > 450 320 550 6w 632

WAVELENGTH ( n m )

9759

0

FIG. 3. Visible/Soret spectra of azide complexes of CcO at partially reduced redox levels. The fully oxidized enzyme at 2.0 mM in sodium phosphate buffer, pH 7.4, was reduced with the appropriate amount of NADH in the presence of 17 p~ phenazine- methosulfate and then treated with sodium azide. The amounts of NADH and azide used were 0.5 and 4.2 mM, respectively, for CcO(III), 1.0 and 4.4 mM, respectively, for CcO(II), and 1.5 and 4.4 mM, respectively, for CcO(1). A, CcO(II1) azide (- - - -) at 7 min after addition of azide and at 60 min after addition of azide (-). B, CcO(I1) azide. C, CcO(1) azide (-) and CcO(0) without azide (- - - -). These spectra were taken for the sample placed in an infrared cell with CaF2 windows and a path length of 0.052 mm.

0.15 N

; 0.10

0.05

Ip m II I 0

0.7 Q) d

0.5 I

a ?

0.3 a d

0. I

FIG. 4. Changes in absorbance at a and Soret band maxima upon progressive reduction of CcO in the presence of azide. The absorbance values for CcO(IV)N3 are taken from the visible/ Soret spectrum of the sample used for the measurement of the infrared spectrum of Fig. 2 and normalized to 2.0 mM. The other values are from Fig. 3.

visible/Soret spectra following azide addition were complete, the infrared spectrum of CcO(III)N3 was measured with the same concentrations of CcO(1V) and azide in the reference cell as were the concentrations of CcO(II1) and azide in the sample cell; 14.3 mM cyanide was also present in the reference cell. The base-line curvature near 2047 cm-' was much greater than in the case of CcO(IV)N3 because of a greater decrease in free azide concentration in the case of CcO(III)N3; addition of extra azide to the sample cell resulted in a flat base line.


For example the addition of 0.8 mM extra azide in the sample cell with 2.0 mM partially reduced enzyme gave an acceptably flat base line (Fig. 5). The amount of extra azide needed in the sample cell was less than the amount of azide bound to the enzyme, because the free azide band in the reference cell was decreased by azide binding to protein even in the presence of cyanide at a concentration high enough to remove all the CcO(IV)N3 bands (see below). The extent of the decrease in intensity due to loss of free azide from protein binding in the reference cell varied somewhat from preparation to preparation. If mismatching of azide concentrations occurred, it was preferable to have a positive peak due to excess free azide in the sample cell, which can be readily identified by deconvolution, than to have a negative peak due to excess free azide in the reference cell. Therefore, for all the infrared measurements of the partially reduced CcO azides, an amount of extra azide appropriate to give a weak positive peak at 2047 cm" was added to the sample cell. The CcO(III)N3 spectrum (Fig. 5) shows three bands, a clearly asymmetric 2040 cm" band and less intense bands at 2016 and 2004 cm". An attempted deconvolution fit with four theoretical curves at 2047, 2039, 2016, and 2004 cm" gave a residual (i.e. the absorbance difference between the experimentally observed spectrum and the sum of the four theoretical curves) with a small peak at 2027 cm" (Fig. 5b) . A flat residual can be achieved with five Gaussian curves (Fig. 5c). The intensities of the 2047 and 2027 cm" bands were less consistent than those for the other bands. The variations in 2027 cm" band intensity from preparation to preparation suggested that this weak band may be due to a contaminant metal azide. However, this band was detected only at this redox level (111). The extensive overlap- ping of bands makes accurate deconvolution difficult. Never- theless the apparent parameters of the 2039, 2016, and 2004 cm" bands were quite consistent. The CcO(I1) and CcO(1) azides gave spectra with three bands clearly evident (Figs. 6

W 0 z Q

LL 0 m

m

m a

, 2047, I 2039

t ,.

I I I I I I 1 2060 2020 I980

WAVENUMBER (cm")

FIG. 5. Infrared spectrum of CcO(1II)Na. a, CcO(II1)azide versus a reference cell containing 2.0 mM Cco(Iv), 14 mM sodium cyanide, and 3.5 mM sodium azide. Contents of the sample cell are as in Fig. 3. b, the residual from the experimental curve (a) less the sum of four theoretical Gaussian curves (the four smooth peaks of a excluding the 2026 cm" peak). c, the residual from the experimental curve in a less all five theoretical curves.

I I I I I

I I 1 I I I

WAVENUMBER (crri') 2060 2040 2020 2000

FIG. 6. Infrared spectrum of CcO(II)N3. a, CcO(I1) azide prepared as in Fig. 3 uersus the same reference cell contents as in Fig. 5. b, the residual from the experimental curve in a less the sum of the three theoretical Gaussian curves shown in a.

I' I I I 1

I ' I I I I 2060 2040 2020 2000

WAVENUMBER (crri')

FIG. 7. Infrared spectrum of CcO(I)Ns. a, CcO(1) azide prepared as in Fig. 3 versus the same reference cell contents as in Fig. 5. b, the residual from the experimental curve in a less the sum of the theoretical curves in a.

and 7). Band parameters for each of the three levels are in Table 11. The 2039 cm" band of CcO(II1) azide of Fig. 5 is identical within experimental accuracy to the 2040 cm" bands in CcO(II)N3 and CcO(I)N3. The distinction between the 2040 cm" band for the partially reduced enzyme azides and the 2041 cm" band of the CcO(1V) azide is clear upon comparing their bandwidths. The intensity of the 2016 cm" band is greatest in CcO(II)N3 and lowest in CcO(III)N3 where it is only about one-half as intense as in CcO(II)N3. The 2040 cm" band is least intense in CcO(I)N3. The 2004 cm" band is seen only in CcO(III)N3. The azide concentrations used here (3.1-4.4 mM) appeared high enough to saturate the metal azide binding sites, since increasing the azide concentration over this range gave no apparent intensification of the relevant infrared bands.

Weak Association of Azide with CcO-Treatment of dithio- nite-reduced CcO(0) with azide caused no changes in visible/ Soret spectrum and resulted in no infrared bands that were consistent with metal azides. However, the IR spectrum meas-

Azide Binding to Iron and Copper Sites in Cytochrome c Oxidase 9761

ured for a mixture of 2.2 mM CcO(0) and 3.4 mM azide in the sample cell, against a reference cell containing the same concentration of a CcO(1V) azide mixture with 14 mM cyanide also present, exhibited a weak, but readily detected, band at 2047 cm" with a Avlh value comparable with the width of a free azide band. This result suggested that a portion of the azide in the reference cell interacts with CcO(1V) even in the presence of cyanide. Subsequent experiments revealed that azide, at high concentrations, binds or associates weakly with CcO at all redox levels. Evidence for such an association is shown in Fig. 8 with an IR spectrum of a mixture of 1.0 mM CcO(I1) and 25.9 mM azide in the sample cell and the same concentration of azide without protein in a variable path length reference cell. The band in Fig. 8 at 2042 cm" cannot be due to mismatching of free azide concentrations in the two cells, because the band maximum is a t lower wavenumber than that of free azide (2047 cm"). The decrease in absorbance of the 2047 cm" band due to the weak association with protein was compensated for by adjusting the pathlength of the reference cell to give the flat base line of Fig. 8. A similar spectrum was readily observed for each of the other redox levels of the enzyme when the azide concentration was greater than 20 mM.

Effects of "N Labeling on Azide IR Spectra-Use of azide terminally labeled with 15N (ie. 15N14N14N) allows more dis- criminating IR characterization of azide bonding to CcO. The "N-labeled azide gives only one band as a free ion in solution but upon bonding to a metal ion gives rise to two bands due to M-''NI4Nl4N and M-'4N14N'6N which exhibit different vN, values (25, 26, 35). For example, as shown in Fig. 9, with CCO(II)'~N'~N"N, five bands can be detected with maxima near 2038, 2033, 2024, 2013, and 1997 cm". Based upon the magnitudes of isotopic shifts obtained with many hemin and Cu(I1) azides, the 2013 and 1997 cm" bands can be reasonably assigned to isotopically shifted bands related to the 2016 cm" band found for the 14N14N14N spectrum (Fig. 6). The weak bands at 2033 and 2024 cm" are similarly related to the 2040 cm" band found for unlabeled azide. The 2038 cm" band of Fig. 8 is at the same wavenumber as is I5NI4Nl4N- free in solution. The effects of 16N on the IR spectra of CcO(1) and CcO(I1) azides are similar. However, the CCO(III)~~N'~N'~N spectrum was more complex with seven bands detected near 2040,2033,2023,2011,1999,1995, and 1985 cm"; the following assignments for 15N14N14N bands related to 14N14N14N bands appear reasonable: 1999 and 1985 cm" bands from the 2004 cm" band, the 2011 and 1995 cm" bands from the 2016 cm" band, the 2033 and 2023 cm" bands from the 2039 cm"

2080 2040 2000 WAVENUMBER (crn")

FIG. 8. Infrared spectrum of azide to non-metal sites in CcO. The sample cell contents were 1.0 mM CcO(I1) and 25.4 mM sodium azide in 50 mM sodium phosphate buffer, pH 7.4. Reference cell contents were 25.4 mM sodium azide without enzyme in a variable path length cell which was adjusted to achieve a flat base line.

I I I , I I I I

I 1 I I I I I 2060 20 20 1980

WAVENUMBER (cni') FIG. 9. Infrared spectrum of CcO(I1) azide with a

terminal16N label in the azide. Conditions are the same as in Fig. 6 except for an enzyme concentration of 2.2 mM and use of 16N'4N'4N azide.

TABLE 111 Effects of "N on azide infrared bands of cytochrome c oxidase azides

at four redox levels Conditions as in Table 11.

Redox level Y (k1.5)

14NI4Nl4N 16N14Nl4N

IV

111

I1

I

Free azide ion

2051 2041 2047 2039 2016 2004 2047 2040 2016 2047 2040 2016 2047

cm"

2039" 2035," 2024" 2040" 2033," 2023" 2011," 1995" 1999," 1985" 2038 2033,2024 2013,1997 2038 2033,2024 2013, 1997 2038

"The accuracies of these Y values are +2-3 cm".

band, and the 2040 cm" band is from the 2047 cm" band. Bands due to isotope splitting of the 2027 cm" band of Fig. 5 were too weak to be positively identified in the CcO(II1) "N14N14N spectrum. In the spectrum of CcO(1V) 15N14N14N three bands were observed at 2039,2035, and 2024 cm". The latter two bands are assignable to those shifted from the 2041 cm" band, whereas the 2039 cm" band must be shifted from 2051 cm" band without splitting. The lack of splitting indicates the lack of strong bonding to the termini of the azide molecules that give rise to the 2051 cm" band. These isotope effects are listed in Table 111.

Effects of CO and CN- on VisiblelSoret and IR Spectra of CcO Azides-When solutions of partially reduced CcO azides under conditions similar to those described in the legend to Fig. 3 were saturated with CO, the visible spectra were con- verted to spectra identical with the carbonyl spectra observed in the absence of azide (13). Furthermore, the IR spectra no longer contained bands at 2016 and 2004 cm", but the 2047 and 2040 cm" bands remained unaffected by the exposure to CO. The displacement of azide from the sites giving rise to the 2016 and 2004 cm" bands by CO was also supported by an increase in the intensity of the 2047 cm-' band which was roughly equivalent to the Fe., concentration. Upon the addition of cyanide at 14.3 mM to solutions of the partially reduced CcO azides under the same conditions as in the exposure to CO, all the azide IR bands, except for the 2047 cm" band, were removed; both IR and visible/Soret spectra in each case


became identical with the corresponding CcO cyanides without azide present. Similarly the two azide bands at 2051 and 2041 cm" for CcO(IV)N3 were removed by 10 mM cyanide. The complete removal of these bands with concomitant appearance of visible/Soret and IR spectra found for CcO(1V)CN required the exposure of the CcO(IV)N3 solution to cyanide for 16 h at 4 "C. The rate of cyanide binding to the enzyme in the presence of azide as followed by visible/Soret spectra was the same as found for cyanide binding in the absence of azide, a finding in conflict with results reported by others (36). The removal of the 2051 and 2041 cm" bands by cyanide provides further evidence that the azide binding sites associated with these bands are at the dioxygen reduction site.

DISCUSSION

Assignment of Infrared Bands-The infrared spectrum for CcO(IV)N3 with bands at 2051 and 2041 cm" provides evidence for azide binding to the fully oxidized enzyme that is strikingly different from the previously held view. Azide has been considered to bind to Fez: based upon indirect evidence largely from EPR and MCD spectra (37, 38). In contrast, our findings suggest that one azide binds to Cug+ to give the 2041 cm" band, one azide associates with protein (presumably near the oxygen reduction site in a manner that does not involve strong bonding to a metal ion) to give the 2051 cm-l band, and an undetermined number of azides, presumably many azides, weakly associated with protein to give a broad band at 2042 cm". Assignment of the 2041 cm" band to an azide bound to a metal is supported by the splitting observed with 15N14N14N-. That the metal is Cug+ is supported by the loss of the 2041 cm" band upon exposure to cyanide with concomitant development of the 2151 cm" band which has been reasonably assigned to CugCN (16). The Cug+N3 assignment is also supported by the infrared spectra for the (I), (11), and (111) levels wherein a similar band can be assigned to Cug+N3 and lower wavenumber bands to Fez:N3 (see below). The very small effects of azide binding on the visible/Soret spectrum (Fig. l), as well as on EPR and MCD spectra (37, 38), are much more consistent with azide binding to Cug+ than to Fe::. The 2051 cm" band is shown not to be due to a metal ion bound azide, since the band is not split by 15N labeling. The much lower AuH for the 2051 cm" band compared with the band for free azide indicates a more rigid and confined environment. Although the location of this environment within the protein is not established, the loss of the 2051 cm" band upon exposure of CcO(IV)N3 to cyanide shows cyanide binding to Cug+ prevents azide from occupying this site and suggests the site is located near CUB and/or Fe,,.

The partially reduced CcO azides exhibit infrared spectra consistent with azide binding to both Cug+ and Fea: and weak associations of azide with protein but no evidence of azide occupying a site comparable to the site represented by the 2051 cm-l band of CcO(IV)N3. The 2040 cm" bands for (I), (11), and (111) level azides can be assigned to Cug+N3 for several reasons. Cyanide removes this band from CcO(III)N3 concomitant with formation of a Cug+CN band at 2151 cm". The vN, of 2040 cm" is comparable with uN, values found for small molecule Cu2+ azides (29-31) and for other Cu2+ protein azides (26). Although the UN, of 2040 cm-l is within the wavenumber range found for high spin hemeprotein azides (25, 39), this band cannot be due to Fe::N3, since exposure to CO results in the full development of a band near 1964 cm" due to Fei: CO without altering the 2040 cm" band. These 2040 cm" bands are also similar in wavenumber to the 2041 cm" band of CcO(IV)N3 which was assigned to Cui+ as discussed

above. However, the partially reduced CcO azide bands are wider than the fully oxidized CcO bands and are, therefore, consistent with a more mobile, less rigid environment for the azide ligand.

The partially reduced CcO azides give bands fully consistent with azide binding in low spin hemin azides. The CcO(1) and CcO(I1) azides exhibit only one such band at 2016 cm", whereas CcO(III)N3 exhibits two bands at 2016 and 2004 cm". These bands disappear concomitantly with the appearance of bands due to Fe::CN or Fe2:CO upon exposure to cyanide or carbon monoxide. Low spin hemin B azides exhibit bands of similar wavenumbers at 2015, 2004, and 2000 cm" with ammonia, pyridine, and N-methylimidazole, respectively, as trans ligands (25, 39). In contrast, high spin hemin B azide gives v N , values of 2059 and 2044 cm" in chloroform and pyridine, respectively (39). Thus the differences in structure that give rise to the two species of CcO(III)N3 represented by bands at 2016 and 2004 cm" may involve different strengths in proximal histidine to Fe,, bonding and/or different environments surrounding the azide ligand. The correla- tion between optical spectra (Fig. 4) and Fea:N3 infrared spectra indicates the structure giving rise to the 2004 cm" band occurs only when heme a is partially oxidized. In CcO(I)N3 and CcO(II)N3 where heme a must be reduced, no 2004 cm" band is seen. In CcO(III)N3 the absorbance difference for the a-peak, i.e. AA602450nm in Fig. 4, which mainly reflects the redox state of cytochrome a, indicates about one- half of cytochrome a is reduced and the infrared spectra show the 2016 and 2004 cm" bands to be of about equal intensity.

The band observed at 2042 cm" at high azide concentrations for CcO at all redox levels represents an unexpected type of bound azide. The lack of effect of overall redox level on this band indicates a redox active metal cannot be involved in this type of azide binding. The high intensity of the band achieved at high azide concentrations is consistent with many molecules of azide binding per molecule of enzyme. The absence of this band at low azide concentrations shows a lower affinity for azide at these sites than at metal sites at most redox levels. Infrared evidence of the interaction of azide with bovine serum albumin has also been observed as a band at 2042 cm" (22). Therefore we conclude that the 2042 cm" band represents azide within a hydrophobic region of the protein. Furthermore the effects of azide on mitochondrial energy conservation may be related to such interactions with F1 of the ATPase and other membrane proteins as well as with CcO (40-44).

Comparison of CcO Azide IR Spectra with Those of Other Hemeprotein Azides-The IR spectra for azide bound to hemin a3 differ in several respects from the spectra for azide bound to hemin B of myoglobins and hemoglobins. Human metHbA and bovine heart metMb azides exhibit bands near 2042,2022, and 2012 cm" (45). The 2042 cm" band has been assigned to the high spin form which with changes in temper- ature, interconverts with the lower spin forms represented by the lower wavenumber bands. The (I), (II), and (111) level CcO azides each exhibit a 2016 cm" Fe3+N3 band and the CcO(III)N3 spectrum also has a band at 2004 cm". No evidence for a high-spin azide band comparable with the bands found in metMb and metHb azides has been found for the CcO azides. The apparent greater destabilization of high spin hemin azide in CcO than in the globins may result from the difference in porphyrin and/or protein structures. With 2,4- disubstituted deuterohemin azides, the more electron-withdrawing are the ring substituents the more the low-spin species are stabilized (27, 39). Thus it is significant that the %formyl group of heme a3 in CcO is much more electron-


withdrawing than is the corresponding 8-methyl of heme B of Mb and Hb (46,47).

The ligand band widths provide information on the mobility or rigidity of the environment surrounding the ligand in different heme proteins. The reduced site, Fez:, for CcO carbonyls is shown by the extraordinarily narrow C-0 stretch bands to be much less mobile and more rigid than the CO binding sites at reduced iron in hemoglobin and myoglobin carbonyls (12-14). In contrast, the oxidized site, FeQ, of CcO does not appear more rigid than the metHb and metMb oxidized iron ligand binding sites since the CN- and NB bands for the three proteins are of comparable width, in some cases even wider for the oxidase than the globins (15, 16). The 0 2

binding site in CcO may become more rigid upon 0 2 binding (14).

Azide Binding and Electron Distribution among Heme and Copper Centers-The azide binding sites and the apparent electron distribution among heme and copper redox centers at five overall redox levels as deduced from the findings of this study are shown in Fig. 10 and compared with results of earlier CN- studies. For CcO(IV), the fully oxidized resting state, exposure to azide results in the binding of azide to Cui+ and also in the weak association of azide with the enzyme, presumably at or near the O2 reduction site, without formation of a typical metal-azide bond. With CcO(III), azide binds to Fei: to form two low spin azides and to Cui+. The Cui+N3 band is wider for CcO(III)N3 than for CcO(IV)N3 as expected for a less rigid ligand environment in CcO(III)N3. The IR bands show both metals of CcO(II1) to which azide binds to be in their higher oxidation state. The visible/Soret spectra indicate heme a is -50% reduced. Since the enzyme is in redox level (111), CuA must also be about 50% Cu+ and 50% Cu2+. The appearance of two Fez:N3 bands at 2016 and 2004 cm-' in CcO(III)N3 may therefore arise from the two species, IIIN3A and IIIN3B, with different electron distribu- tions between Fe. and CuA as shown in Fig. 10. At level (11), azide also binds to Fei: and Cui+ leaving both Fe. and CuA obligatorily reduced. The addition of another electron equivalent to CcO(I1) results in azide bands at 2016 cm-' for Fe::N3 and at 2040 cm-' for Cu2+N3 that are significantly decreased in intensity without any changes in UN, and Aulh (Table 11). The one-electron reduction of CcO(I1) azide also caused changes in the Soret band that are consistent with about 50% of heme a3 becoming reduced (Fig. 4). This conclusion is based on the interpretation that most of the changes in both visible and Soret regions shown in Fig. 4 upon reduction from (IV) to (11) are due to reduction of Fei+ to Fez+ (4). The changes in Soret band intensity from (11) to (0) is, therefore, due to reduction of Fell to Fef:. These results for CcO(I)N3 are consistent with azide binding to Fea3 and CUB only when these metals are in their higher oxidation states and with the fact that in redox level (I) for any given CcO monomer only one of the two metals can be at the higher oxidation state. Con- sequently there will be a mixture of monoazides, one with azide bound only to Fei: with the other three metals at lower oxidation states and another monoazide with azide at Cui+ and the other metals reduced as shown in Fig. 10. An alternative explanation for azide binding in CcO(I)N3 is one where CcO(I)N3 is actually a mixture of CcO(II)N3 and CcO(0). However, this alternative is unlikely because the intensity and the position of the a-band of CcO(I)N3 are almost identical to those of CcO(II)N3 but distinctly different from those of CcO(0). Furthermore the entire spectrum of CcO(I)N3 in the visible region is distinctly different from the average spectrum computedfrom CcO(II)N3 and CcO(0) spectra. Thus the visible spectra confirm the presence of three-electron-

reduced CcO. These findings suggest that the infrared band for Fez:N3 is not influenced by the oxidation state and/or state of ligation of Cug.

Ligand Binding and Interactions Between Redox Centers- The direct evidence of ligand binding to Fe., and/or CUB ions obtained from ligand infrared spectra provides new insights into the accessibility of Fea3 and CUB to ligands at different overall redox levels of the enzyme, the effects of ligands on the electron distribution among redox centers, and the degree of interdependence among these centers. CO forms stable bonds only to Fez:. CO cannot bind to CcO(1V) but does form FeZlCO at any redox level from (111) to (0) (13). Thus CO can recruit an electron to the Fe, site at the expense of the other redox active metals. For example, the bonding of CO to Fe. as Fez+ forces the other metals to be oxidized, i.e. Fe::, Cui+, and Cui+, a condition supported by visible/Soret spectra (13). In contrast, CN- may bind to Fe, and CUB in either oxidized or reduced state (Fig. 10). Infrared spectra support CN- binding only to Cui+ in CcO(1V)CN but indicate CN- binds to Fe:: at levels (111), (11), and (I), to Feq: only at (0), to Cug a t (IV) and (111), and to Cug at (II), (I), and (0). Thus CN- recruits an electron to CUB at levels (11) and (I), but not at (111), and forms Fe2+CN only when Fe., must be reduced as in CcO(0)CN. Upon exposure of CcO cyanides to CO at redox level (0), but not at any other redox level, Fe2:CO forms at the expense of FezlCN. Thus CO effectively forms stable Fe2:CO bonding in competition with CN-.

Azide binding differs from the binding of either CO or CN-. There is no evidence of azide binding to reduced metal ions. Azide binds to Cup at each of four redox levels, (IV) to (I), but binds to Fe:: only in the partially reduced species (I) to (111) (Fig. 10). Azide repels an electron from the metal ion it binds to, whereas CO recruits an electron to the metal ion to which it binds. When CcO azides (I), (11), or (111) are exposed to CO, CO binds to Fez: displacing N: and recruiting an electron to Fee, without affecting N: binding at Cui+N3. On the other hand CN- readily displaces N; from both Fe,, and CUB at all redox levels. With CcO(IV)N3 not only is the N, at Cug+ displaced by CN- but also displaced is an azide that is not bound to a metal ion but may be at or near the O2 reduction site as discussed above.

In summary, the apparent relative affinities of these ligands are CO > CN- >> N: for Fez:, CN- > N; >> CO for FeC, CN- > CO >> N: for Cui, and CN- > N; >> CO for Cui+.

The ligand infrared spectra reveal no interactions between the Fe, and CUB ligand binding sites at all redox levels except CcO(IV), wherein an as yet unestablished bridge between Fei: and Cu&+, which is represented by X in Fig. 10 and is possibly sulfur or chlorine, has been suggested to mediate electromagnetic coupling between the two paramagnetic metal ions (18, 37). The resting oxidized enzyme has been widely studied, since it is the most readily accessible form. Never- theless it is well to note that the resting fully oxidized structure is not likely to participate in oxidase function under turnover conditions (48, 49). There is strong evidence for the binding of CN- to CcO(1V) converting hemin a3 from a high- spin to a low-spin species. This change of spin state was initially ascribed to an Fei:CN structure. More recent evidence made a l-cyano structure, i.e. Fei:CNCui+, more at- tractive (38, 50). However, cyanide infrared spectra have not revealed either a bridging CN- or a CN- bound solely to Fei:, but do support CN- binding only to Cug+ (16). We suggested that CN- binding to Cug+ alters the bonding between X and CUB which affects the bonding between X and Fea3 in such a way as to result in low spin heme a3 (16) and that this change in Fe,, coordination chemistry causes changes


r

+N; - r 0 I

C N

I 0 I I I -CN ItI

L """"_ A

N N

Fe,-- - + N j

Fea--"CuA - 0

C N

C N N'N,N N-N.N

r

& l e -

+N; - I . N 3

N

+ N; ,-b no metal- bound azide

c N N

FIG. 10. Schematic representation of proposed electron distribution and sites of azide and cyanide binding for cytochrome c oxidase at five redox levels.

in visible/Soret and MCD spectra (50,51) that are consistent with a high to low spin state conversion. The azide infrared spectra reported here shows that N; also binds to Cug+ of CcO(1V) but, in contrast to CN-, the binding of N: does not significantly affect heme a3 so that the changes in visible/ Soret (Fig. 1) and MCD spectra (38,50) are very small. Clearly the binding of N; to Cug+ does not affect X bonding to Fel: in a way that converts heme a3 to a low-spin structure. If N; did bind directly to Fell, then heme a3 is expected to become low-spin, since infrared spectra show that, when N; binds to Fei: in partially reduced CcO species, a low-spin hemin azide results. The observed sensitivity of the Fe:: structure to CN- binding at Cui+ but not to N; binding is consistent with

cyanide being a more nucleophilic anion than azide and with cyanide binding to Cug+ with greater affinity.

A striking independence of Fe, and CUB ligand binding sites in partially and fully reduced CcO carbonyls, cyanides, and azides is demonstrated by the insensitivity of infrared band parameters for ligands bound to one metal to changes in structure at the other metal (13, 16). This independence may be considered surprising in view of the earlier evidence for electromagnetic interactions between these two metals in CcO(1V) as discussed above (37) and the mechanistic propos- als for formation of an intermediate with a p-peroxo bridge between Fez: and Cug+ during O2 reduction to water (2, 12, 19). This ligand binding site independence at the O2 reduction


site was first demonstrated by the very small effects of overall redox level on the C-0 stretch band parameters upon reduction of CcO(1II)CO to CcO(0)CO (13, 14). The very small effects of changes in oxidation state at the other redox centers on the C-0 infrared band were unexpected since Fe,, and CUB are expected to be within 5 A of each other and the carbonyl spectra of heme and other hemeprotein carbonyls show a large sensitivity to changes in the trans ligand and the CO ligand environment (solvation and steric effects) (25, 52). Further- more, the presence or absence of either CN- or N; at CUB has little or no effect on the CO band at Fe:: at various overall redox levels (16). Conversely, the infrared bands of CN- or N; at CuB are insensitive to either the ligand bound to Fe:: or to the oxidation state of Fe,,.

The insensitivity of a ligand infrared band at one metal to changes in ligand and/or oxidation state at the other metal restricts the possible stereochemical arrangements of Ligands at Fe., and CU,. For example, if CUB is less than 5 A from Fe.,, then it seems sterically impossible for ligands such as CO, CN-, and N, to occupy binding sites at both metals such that the two ligands are pointing towards each other (i .e. in the steric orientation required for formation of a p-peroxo linkage, Fe3+-0-0-Cu", during the reduction of O2 to water) without the ligands perturbing each others infrared spectrum. Clearly ligand binding and/or oxidation state changes at one metal must occur without significantly affecting either the bonding or the environment of the ligand at the second metal if the parameters of the ligand infrared band for the second metal are to remain unchanged. Ligand infrared spectra thus provide a uniquely useful means of detecting perturbations of one site on another. The observed independence of Fe,, and Cue sites with azide provides further support for the O2 reduction mechanism we proposed earlier (14, 16) with Fei:OOH, rather than Fe:,:OOCug+, as an intermediate. Re- cent Raman evidence gives direct evidence for the hydroper- oxide intermediate (53).

The appearance of two low-spin hemin azide bands at 2016 and 2004 cm" in CcO(III)N3, but only one such band at 2016 cm" for CcO(II)N3 and CcO(I)N3 can be considered evidence of either heme a to heme a3 or copper A to heme u3 interaction in CcO(III)N3. As shown in Fig. 10, CcO(III)N3 may be represented as two species, one ( A ) with Fe:' and Cuy and the other ( B ) with Fei+ and Cui; both Fe,, and Cue are shown to be oxidized by the azide infrared spectra. The 2016 cm" band of CcO(III)N3 can therefore be the form with either Fe, or CUA reduced in analogy to the 2016 cm-' band of CcO(II)N3 which has Fe:+ and Cui. In other words, either FeF or Cui, but only one of the two, may stabilize the 2016 cm" band structure. It is equally possible that one of the oxidized metal ions (Fez+ or Cui+) induces the structure associated with the 2004 cm-' band. Evidence less directly related to structural changes, mainly from redox titrations in the presence or absence of inhibitors, may also support such heme-heme interactions (54-56). The interactions must be long range since the effector metal, whether Fe, or CUA, is expected to be at least 12 A away from FeB, (57,58). It is of interest that the infrared data give no evidence of similar long range effects of Fe, or CUA oxidation state on the ligand binding site of CuB. The shift in v,, from 2016 to 2004 cm-' with a change in oxidation state at Fe, or CuA could result from a change in protein conformation that increases the strength of the proximal histidine to FeB, bond, alters the environment about the N; ligand in a solvation and/or steric sense, and/or affects porphyrin A at farnesylethyl, formyl, or other portions of the molecule (46). Distinguishing between these possible types of

long distance effect poses an intriguing experimental chal- lenge for the future. We had previously found experimental support for similar heme-heme interactions in the C-0 infrared spectra of CcO carbonyls at different redox levels (13). Thus the infrared spectra of both N; and CO provide direct evidence of significant alterations in the binding of these ligands at the O2 reduction site as a consequence of changes in the oxidation state at a remote metal center. Recent inter- nal electron transfer studies (59) and observations of an effect of CN- or CO binding to Fe., on the Soret spectrum of reduced heme a (60) also appear consistent with the long range interactions demonstrated by the infrared studies. The conforma- tional changes associated with electron transfers are expected to make a critical contribution to both respiratory control and the coupling between electron transfer and proton transloca- tion.

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Infrared Evidence of Azide Binding to Iron, Copper, and Non-metal