Complex Formation Between the Copper Protein, Azurin and the Cytochrome c Peroxidase of Pseudomonas Aeruginosa Thomas Brittain and Colin Greenwood TB. Department
of Biochemistry, University of Auckland, Aucklarul, New Zealand.-CG. School of Biological Sciences, University of East Anglia, Norwich, U.K.
ABSTRACT Reduced azurin reacts with the resting, oxidized cytochrome c peroxidase of Pseudomonas aeruginosa to yield time courses observed at 420 nm, which consist of the sum of two exponential processes. Each process exhibits a hyperbolic dependence of the observed rate constant on the reduced azurin concentration. The fraction of the total optical density change which each process contributes is found to be dependent on the reduced azurin concentration. This pattern of reactivity is maintained at pH values between 5.5 and 8.0. The data has been analyzed in terms of a complex formation between the two proteins followed by an intramolecular electron exchange reaction. This analysis yields values for the binding constants at each pH value. The intramolecular exchange reaction is independent of pH, whilst the pH dependence of the binding reaction suggests the involvement of a histidine residue in this process.
INTRODUCTION The Gram negative bacterium Pseudomonas aeruginosa produces cytochrome c peroxidase [ll which catalyzes the reaction :
a dihaem
Electron donor + Hydrogen peroxide s Water + Oxidized donor. The enzyme has a molecular weight of 44,000 [2,31 and contains two nonequivalent, covalently bound c type haems [4] with redox potentials of +320 mV and -330 mV, respectively [Sl. The enzyme can employ either cytochrome cS5r or the blue copper protein, azurin as electron donor [6]. Over the years a great deal of work has been carried out isolating and characterizing the various forms of
Address reprint requests and correspondence to: Professor C. Greenwood, Sciences, University of East Anglia, Norwich, U.K. Journal of Inorganic Biochemistry, 48,71-77 (1992)
School of Biological
71 0 1992 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, NY, NY 10010 0162-0134/92/$5.00
72
T. Brittain and C. Greenwood
the enzyme present in the catalytic cycle [7-B]. Although a number of investigations have appeared concerning various parts of the catalytic cycle and some elementary steps have been identified in the reaction scheme [12, 16, 171,it is only recently that a full quantitative mechanism has been proposed and tested against experimental measurements [18]. Of particular interest is the fact that part of the overall reaction process can be isolated and investigated by reacting the dochrome c, peroxidase with reduced &rin in the absence-of the second substrate, hydrogen peroxide [18]. Under these conditions the cytochrome c peroxidase undergoes single electron reduction of the high potential haem to yield the half-reduced form of the enzyme. A quantitative analysis of this reaction at a single pH value has appeared [18] and is consistent with ‘the following reaction scheme:
Peep + AZ’+
[Peep- At “I
-
Half reduced Peep
“2
3+
+
PCCD.
+
AZ’+
(W-AZ
,
AZ-
’ +j
k2 SCHEME 1.
where Peep and Peep represent two reactive forms of the enzyme in equilibrium and {Peep-AZ}r@i&ents an intermediate complex formed between the two proteins [18]. In this paper we report the results of a series of experiments performed over a range of pH values, aimed at identifying possible amino acids which might play a role in controlling the complex formation process and the subsequent intramolecular electron transfer reaction. ExPERIn4ENTAL Pseuabmonas aen@nosa was grown in 100 1 cultures and both the cytochrome c
peroxidase and axurin were purified from the resulting cells as described earlier [lo, 191.Cytochrome c peroxidase used in these studies had an A,,/A,,, purity ratio of 4.65. The concentration of cytochrome c peroxidase and azurin were determined by absorption measurements on the oxidized forms of the proteins at 407 nm and 625 nm, employing extinction coefficients of 237 m&I-’ cm-’ and 5.1 mM-’ cm-‘, respectively [20, 211.Axurin was reduced by the addition of a
AZURIN-CYTOCHROME
c PEROXIDASE
COMPLEX
73
small excess of sodium dithionite followed immediately by passage down a 25 cm x 1 cm column of Sephadex G25, equilibrated with the appropriate buffer. All reactions were performed anaerobically under an atmosphere of oxygen free nitrogen. In all cases the buffers used were 25 mM sodium phosphate at 25°C. Reactions were monitored at 420 nm using a Gibson and Milnes stopped flow apparatus as previously described 1221.Reaction time courses were deconvoluted into the sum of two exponentials using standard graphical techniques. The dependences of the observed rate constants on azurin concentration were found to hyperbolic and were fitted to hyperbolic function using a nonlinear leastsquares computer algorithm developed in this laboratory. RESULTS AND DISCUSSION When reduced azurin was rapidly mixed with oxidized cytochrome c peroxidase and the reaction was monitored at 420 nm the time course followed a double exponential curve (Fig. 1). These time courses could easily be deconvoluted to yield two observed rate constants (fast and slow). At a single pH value the observed rate constants for both the fast and slow processes followed a hyperbolic concentration dependence. Furthermore, the dependence on reduced azurin concentration was found to be itself dependent on the pH at which the reactions were performed (Fig. 2). Double reciprocal plots of the pH data of Figure 2 showed that although the gradients of the plots were pH dependent the intercept on the l/k axis was not altered by pH (Fig. 3a, b). These findings are consistent with the model presented in Scheme 11181, if the rate of intramolecular electron transfer (k, and k_,) is not altered by solution pH whilst the complex formation reaction is. According to the analyses previously presented [23], at any particular pH value each of the two forms of the cytochrome c peroxidase present in the scheme should exhibit a hyperbolic dependence of observed rate constant on reduced azurin concentration given by:
WWt~‘+
1
1 + (k,/k,)[Az’+]
as is indeed seen in Figure 2. In this scheme the intramolecular electron transfer process within the complex has a rate of k, and represents the limiting rate at high azurin concentrations. The ratio (k/k,) represents the biding constant for the complex. The data of Figure 3 show that the limiting rate (k,) at high azurin concentrations is not pH dependent. Values for the binding constants for both forms of the cytochrome c peroxidase at each pH value were obtained using a
FIGURE
1. The time conrse of the reaction of 2 PM cytochrome c peroxidase with -80 mM reduced azurin follows at 420 nm. The reaction was performed in 25 mM sodium phosphate buffer at 25 “C, pH 8.0.
14
T. Brittain and C. Greenwood
[A?]
ml-l
(a)
FIGURE 2. The concentration dependence of the observed rate constants for the fast (a) and slow (b) reactions of cytochrome c peroxidase and reduced azurin. The lines represent nonlinear leastsquares best fit data to a hyperbola for data obtained at pH 5.5 (Cl), pH 6.0 (m), pH 6.6 (A), ph 7.0 (01, pH 7.5 (O), and pH 8.0 ( v 1.
nonlinear least-squares fitting procedure (Fig. 4). These binding constants show a pH dependence which follows a titration curve with an associated pK value of approximately 7.0. Such a pK is strongly suggestive of the involvement of a His residue in the process of complex formation. The question then arises as to whether the important His residue is present in the azurin molecule or the cytochrome c peroxidase molecule. Recent studies on other electron transfer reactions involving reduced azurin suggest the most likely answer is that the His residue controlling complex formation in this study is one present on the surface of the azurin molecule. Although two distinct surface areas on the azurin molecule have been implicated in electron transfer reaction [24, 241, namely the “His patch”, which represents a surface area centered on His35, and the “hydrophobic patch” surrounding Met 44, only the hydrophobic patch appears to be directly involved. The hydrophobic patch which includes the partially exposed copper ligand residue His,,, has been shown by site directed mutagenesis to be involved in the self-exchange reaction of azurin 126-281 in the reaction between azurin and nitrite reductase and with cytochrome cS5, 1291.We would therefore speculate that this residue, namely HisIr,, is the most likely candidate for that residue controlling the pH dependence of the complex formation equilibrium constant.
AZURIN-CYTOCHROME
c PEROXIDASE
COMPLEX
75
24 '/kt
1.6
I
.02
*01
.06
'4d'l (a)
60
V
l/k 5 40
.02
.01
FIGURE 3. Double reciprocal plots of the fast (a) and slow (b) data of Figure 2.
%A$+1
0)
From the pH dependence of the rate constants to the effect of pH on the proportions of the two reactive forms of cytochrome c peroxidase present in solution we have observed a pH dependence in this parameter (Fig. 5). As shown previously 1181,at any particular pH value Scheme 1 accurately predicts the observed concentration dependence of the contribution to the overall optical density change corresponding to the fast reaction. However, from Figure
160\w 120.
6.0
7.0 PH
80
FIGURE 4. The pH dependence of the apparent binding constant for the fast (W) and slow (0 1 kinetic processes obtained from the hyperbolic data of Figure 2.
76
T. Brittainand C. Greenwood
FIGURE 5. The concentration dependence of the proportion of the overall optical density change observed at 420 nm, contributed by the fast reaction at pH 5.5 (01, pH 6.5 (A 1, and ph 8.0 (v 1. Other curves have been omitted for clarity. 5 it is apparent that this parameter is also pH dependent and titrates in the region of pH 7.0. The origin of this effect must arise from a pH linked change in the proportions of the two reactive forms of the cytochrome c peroxidase present at equilibrium in solution (Peep and Peep of Scheme 1). It has previously been argued ‘that this equilibrium is associated with the measured spin equilibrium of the high potential haem [lo, 11, 181. If this is the case then our data would predict a pH dependence of this spin equilibrium. Based on amino acid sequence data and comparison with the known three-dimensional structures of cytochrome c2 and cytochrome c [31], it has been suggested that the haem-haem interactions between the two physically close haems of cytochrome c peroxidase [15, 301 which controls the spin equilibrium is mediated by His,,. It is tempting to suggest the pH dependence observed in Figure 5 and the concomitant shift in spin state of the high potential haem arises from the ionization of residue His,,. Clearly, the exact identification of which of the amino acids outlined above is responsible for the observed pH dependences is speculative and confirmation must await the availability of specific site directed mutants such as those recently used in some other studies (32).
We gratefully acknowledge the support of the S.E.RC. (UK) in this work and the expert technical assistance of Mr. R Little.
REFERENCES 1. 2. 3. 4. 5.
N. Ellfolk and R. Soininen, Acta Chem. Stand. 24, 2126 (1970). R. Soininen, N. Ellfolk, and N. Kalkinen, Acra Gem. Scand. 27,1106 (1973). R. Soininen and N. Ellfolk, Acta Gem. Scar&f.27,2193 (1973). R. Soininen, H. Sojonen, and N. Ellfolk, Acta Chem. Scand. 24,2314 (1970). N. Elifolk, M. Ronnberg, R. Aasa, L. E. Andreason, and T. Vanngard, Biochim. Biophys. Acta 743,23 (1983). 6. N. Ellfolk, M. ROMberg, R. Aasa, L. E. Andreason, and T. Vanngard, Biochirn. Biophys. Acta 784,62 (1984). 7. C. Greenwood, N. Foote, J. Petersen, and A. J. Thomson, Bbchemm J. 223, 379 (1984). 8. N. Foote, J. Petersen, P. M. A. Gadsby, C. Greenwood, and A. J. Thomson, Biochem. J. 223,369 (1984).
AZURIN-CYTOCHROME
c PEROXIDASE COMPLEX
9. C. Greenwood,
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
77
N. Foote, J. Petersen, and A. J. Thomson, Biochem. Sk Tmns. 13, 625 (1985). N. Foote, J. Petersen, P. M. A. Gadsby, C. Greenwood, and A. J. Thomson, Biochem. Z. 230,227 (1985). C. Greenwood, N. Foote, P. M. A. Gadsby, and A. J. Thomson, Chimicu. Sctipta. 28A, 79 (1988). M. Ron&erg, N. Ellfolk, and H. B. Dunford, Acta Chem. Scund. B38,79 (19841. R. Aasa, N. Ellfolk, M. Ronnberg, and T. Vanngard, FEBS Let&. 250,175 (1989). M. Ron&erg, K. Qsterlund, and N. Ellfolk, Biochim. Biophys. Acta 670, 170 (1981). N. ElIfolk, M. Ron&erg, R. Aasa, T. Vanngard, and J. Angstrom, Biochim. Biophys. Acra 791, 9 (1984). M. Ron&erg, A. M. Lambier, N. Ellfolk, and H. B. Dunford, Arch. Biuchem. Bicphys. 236,714 (1985). M. Ronnberg, T. Araison, N. Ellfolk, and H. B. Dunford, J. Biol. Chem 256, 2471 (1981). N. Foote, R. Turner, T. Brittain, and C. Greenwood, Biochem J. (in press). N. Foote, A. J. Thomson, D. Barber, and C. Greenwood, Bbchem J.2@9,701(1983). R. Soininen and N. Ellfoik, Acfa Chem. Scund. 26,861 09721. R. Soininen and N. ElIfolk, Acta Chem. Scat&. 27,35 (19731. Q. H. Gibson and L Mimes, Biochem. J 91,161(1964). S. Strickland, G. Palmer, and V. Massey, Z. Biol. Gem. 250,4048 ( 1975). 0. Farver and I. Pecht, Zsmel. J. Chem:21, 13 (1981). 0. Farver, Y. Blatt, and I. Pecht, Biochemistty 21,3556 (1982). M. van de Ramp, R. Floris, F. C. Hali, and G. W. Canters, J. Amer. Chem. Sot. 112, 907 (1990). C. M. Groenveld and G. W. Canters, Eur. J. Biochem. 153,559 (1985). C. M. Groenveld and G. W. Canters, Z. Biol. Gem. 263,167 (1988). M. van de Ramp, M. C. Silvestrini, M. Brunori, J. V. Beeuman, F. C. Hali, and G. W. Canters, Eur. J. Biochem. 194, 109 (1990). R. Aasa, N. Ellfoik, M. Ron&erg, and T. Vanngard, Biochim. Biophys. Acta 670,170 (1981). N. Ellfolk, M. Ron&erg, and K. Osterlund, Biochim. Biophys. Actu 1080, 68 (1991). M. van de Ramp, F. C. Hali, N. Rosa@ A. Finazzi-Agro, and G. W. Canters, B&him. Biophys. Acta 1019,283 (1990).
Received December 23, 1991; accepted March 3, 1992
of Biochemistry, University of Auckland, Aucklarul, New Zealand.-CG. School of Biological Sciences, University of East Anglia, Norwich, U.K.
ABSTRACT Reduced azurin reacts with the resting, oxidized cytochrome c peroxidase of Pseudomonas aeruginosa to yield time courses observed at 420 nm, which consist of the sum of two exponential processes. Each process exhibits a hyperbolic dependence of the observed rate constant on the reduced azurin concentration. The fraction of the total optical density change which each process contributes is found to be dependent on the reduced azurin concentration. This pattern of reactivity is maintained at pH values between 5.5 and 8.0. The data has been analyzed in terms of a complex formation between the two proteins followed by an intramolecular electron exchange reaction. This analysis yields values for the binding constants at each pH value. The intramolecular exchange reaction is independent of pH, whilst the pH dependence of the binding reaction suggests the involvement of a histidine residue in this process.
INTRODUCTION The Gram negative bacterium Pseudomonas aeruginosa produces cytochrome c peroxidase [ll which catalyzes the reaction :
a dihaem
Electron donor + Hydrogen peroxide s Water + Oxidized donor. The enzyme has a molecular weight of 44,000 [2,31 and contains two nonequivalent, covalently bound c type haems [4] with redox potentials of +320 mV and -330 mV, respectively [Sl. The enzyme can employ either cytochrome cS5r or the blue copper protein, azurin as electron donor [6]. Over the years a great deal of work has been carried out isolating and characterizing the various forms of
Address reprint requests and correspondence to: Professor C. Greenwood, Sciences, University of East Anglia, Norwich, U.K. Journal of Inorganic Biochemistry, 48,71-77 (1992)
School of Biological
71 0 1992 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, NY, NY 10010 0162-0134/92/$5.00
72
T. Brittain and C. Greenwood
the enzyme present in the catalytic cycle [7-B]. Although a number of investigations have appeared concerning various parts of the catalytic cycle and some elementary steps have been identified in the reaction scheme [12, 16, 171,it is only recently that a full quantitative mechanism has been proposed and tested against experimental measurements [18]. Of particular interest is the fact that part of the overall reaction process can be isolated and investigated by reacting the dochrome c, peroxidase with reduced &rin in the absence-of the second substrate, hydrogen peroxide [18]. Under these conditions the cytochrome c peroxidase undergoes single electron reduction of the high potential haem to yield the half-reduced form of the enzyme. A quantitative analysis of this reaction at a single pH value has appeared [18] and is consistent with ‘the following reaction scheme:
Peep + AZ’+
[Peep- At “I
-
Half reduced Peep
“2
3+
+
PCCD.
+
AZ’+
(W-AZ
,
AZ-
’ +j
k2 SCHEME 1.
where Peep and Peep represent two reactive forms of the enzyme in equilibrium and {Peep-AZ}r@i&ents an intermediate complex formed between the two proteins [18]. In this paper we report the results of a series of experiments performed over a range of pH values, aimed at identifying possible amino acids which might play a role in controlling the complex formation process and the subsequent intramolecular electron transfer reaction. ExPERIn4ENTAL Pseuabmonas aen@nosa was grown in 100 1 cultures and both the cytochrome c
peroxidase and axurin were purified from the resulting cells as described earlier [lo, 191.Cytochrome c peroxidase used in these studies had an A,,/A,,, purity ratio of 4.65. The concentration of cytochrome c peroxidase and azurin were determined by absorption measurements on the oxidized forms of the proteins at 407 nm and 625 nm, employing extinction coefficients of 237 m&I-’ cm-’ and 5.1 mM-’ cm-‘, respectively [20, 211.Axurin was reduced by the addition of a
AZURIN-CYTOCHROME
c PEROXIDASE
COMPLEX
73
small excess of sodium dithionite followed immediately by passage down a 25 cm x 1 cm column of Sephadex G25, equilibrated with the appropriate buffer. All reactions were performed anaerobically under an atmosphere of oxygen free nitrogen. In all cases the buffers used were 25 mM sodium phosphate at 25°C. Reactions were monitored at 420 nm using a Gibson and Milnes stopped flow apparatus as previously described 1221.Reaction time courses were deconvoluted into the sum of two exponentials using standard graphical techniques. The dependences of the observed rate constants on azurin concentration were found to hyperbolic and were fitted to hyperbolic function using a nonlinear leastsquares computer algorithm developed in this laboratory. RESULTS AND DISCUSSION When reduced azurin was rapidly mixed with oxidized cytochrome c peroxidase and the reaction was monitored at 420 nm the time course followed a double exponential curve (Fig. 1). These time courses could easily be deconvoluted to yield two observed rate constants (fast and slow). At a single pH value the observed rate constants for both the fast and slow processes followed a hyperbolic concentration dependence. Furthermore, the dependence on reduced azurin concentration was found to be itself dependent on the pH at which the reactions were performed (Fig. 2). Double reciprocal plots of the pH data of Figure 2 showed that although the gradients of the plots were pH dependent the intercept on the l/k axis was not altered by pH (Fig. 3a, b). These findings are consistent with the model presented in Scheme 11181, if the rate of intramolecular electron transfer (k, and k_,) is not altered by solution pH whilst the complex formation reaction is. According to the analyses previously presented [23], at any particular pH value each of the two forms of the cytochrome c peroxidase present in the scheme should exhibit a hyperbolic dependence of observed rate constant on reduced azurin concentration given by:
WWt~‘+
1
1 + (k,/k,)[Az’+]
as is indeed seen in Figure 2. In this scheme the intramolecular electron transfer process within the complex has a rate of k, and represents the limiting rate at high azurin concentrations. The ratio (k/k,) represents the biding constant for the complex. The data of Figure 3 show that the limiting rate (k,) at high azurin concentrations is not pH dependent. Values for the binding constants for both forms of the cytochrome c peroxidase at each pH value were obtained using a
FIGURE
1. The time conrse of the reaction of 2 PM cytochrome c peroxidase with -80 mM reduced azurin follows at 420 nm. The reaction was performed in 25 mM sodium phosphate buffer at 25 “C, pH 8.0.
14
T. Brittain and C. Greenwood
[A?]
ml-l
(a)
FIGURE 2. The concentration dependence of the observed rate constants for the fast (a) and slow (b) reactions of cytochrome c peroxidase and reduced azurin. The lines represent nonlinear leastsquares best fit data to a hyperbola for data obtained at pH 5.5 (Cl), pH 6.0 (m), pH 6.6 (A), ph 7.0 (01, pH 7.5 (O), and pH 8.0 ( v 1.
nonlinear least-squares fitting procedure (Fig. 4). These binding constants show a pH dependence which follows a titration curve with an associated pK value of approximately 7.0. Such a pK is strongly suggestive of the involvement of a His residue in the process of complex formation. The question then arises as to whether the important His residue is present in the azurin molecule or the cytochrome c peroxidase molecule. Recent studies on other electron transfer reactions involving reduced azurin suggest the most likely answer is that the His residue controlling complex formation in this study is one present on the surface of the azurin molecule. Although two distinct surface areas on the azurin molecule have been implicated in electron transfer reaction [24, 241, namely the “His patch”, which represents a surface area centered on His35, and the “hydrophobic patch” surrounding Met 44, only the hydrophobic patch appears to be directly involved. The hydrophobic patch which includes the partially exposed copper ligand residue His,,, has been shown by site directed mutagenesis to be involved in the self-exchange reaction of azurin 126-281 in the reaction between azurin and nitrite reductase and with cytochrome cS5, 1291.We would therefore speculate that this residue, namely HisIr,, is the most likely candidate for that residue controlling the pH dependence of the complex formation equilibrium constant.
AZURIN-CYTOCHROME
c PEROXIDASE
COMPLEX
75
24 '/kt
1.6
I
.02
*01
.06
'4d'l (a)
60
V
l/k 5 40
.02
.01
FIGURE 3. Double reciprocal plots of the fast (a) and slow (b) data of Figure 2.
%A$+1
0)
From the pH dependence of the rate constants to the effect of pH on the proportions of the two reactive forms of cytochrome c peroxidase present in solution we have observed a pH dependence in this parameter (Fig. 5). As shown previously 1181,at any particular pH value Scheme 1 accurately predicts the observed concentration dependence of the contribution to the overall optical density change corresponding to the fast reaction. However, from Figure
160\w 120.
6.0
7.0 PH
80
FIGURE 4. The pH dependence of the apparent binding constant for the fast (W) and slow (0 1 kinetic processes obtained from the hyperbolic data of Figure 2.
76
T. Brittainand C. Greenwood
FIGURE 5. The concentration dependence of the proportion of the overall optical density change observed at 420 nm, contributed by the fast reaction at pH 5.5 (01, pH 6.5 (A 1, and ph 8.0 (v 1. Other curves have been omitted for clarity. 5 it is apparent that this parameter is also pH dependent and titrates in the region of pH 7.0. The origin of this effect must arise from a pH linked change in the proportions of the two reactive forms of the cytochrome c peroxidase present at equilibrium in solution (Peep and Peep of Scheme 1). It has previously been argued ‘that this equilibrium is associated with the measured spin equilibrium of the high potential haem [lo, 11, 181. If this is the case then our data would predict a pH dependence of this spin equilibrium. Based on amino acid sequence data and comparison with the known three-dimensional structures of cytochrome c2 and cytochrome c [31], it has been suggested that the haem-haem interactions between the two physically close haems of cytochrome c peroxidase [15, 301 which controls the spin equilibrium is mediated by His,,. It is tempting to suggest the pH dependence observed in Figure 5 and the concomitant shift in spin state of the high potential haem arises from the ionization of residue His,,. Clearly, the exact identification of which of the amino acids outlined above is responsible for the observed pH dependences is speculative and confirmation must await the availability of specific site directed mutants such as those recently used in some other studies (32).
We gratefully acknowledge the support of the S.E.RC. (UK) in this work and the expert technical assistance of Mr. R Little.
REFERENCES 1. 2. 3. 4. 5.
N. Ellfolk and R. Soininen, Acta Chem. Stand. 24, 2126 (1970). R. Soininen, N. Ellfolk, and N. Kalkinen, Acra Gem. Scand. 27,1106 (1973). R. Soininen and N. Ellfolk, Acta Gem. Scar&f.27,2193 (1973). R. Soininen, H. Sojonen, and N. Ellfolk, Acta Chem. Scand. 24,2314 (1970). N. Elifolk, M. Ronnberg, R. Aasa, L. E. Andreason, and T. Vanngard, Biochim. Biophys. Acta 743,23 (1983). 6. N. Ellfolk, M. ROMberg, R. Aasa, L. E. Andreason, and T. Vanngard, Biochirn. Biophys. Acta 784,62 (1984). 7. C. Greenwood, N. Foote, J. Petersen, and A. J. Thomson, Bbchemm J. 223, 379 (1984). 8. N. Foote, J. Petersen, P. M. A. Gadsby, C. Greenwood, and A. J. Thomson, Biochem. J. 223,369 (1984).
AZURIN-CYTOCHROME
c PEROXIDASE COMPLEX
9. C. Greenwood,
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
77
N. Foote, J. Petersen, and A. J. Thomson, Biochem. Sk Tmns. 13, 625 (1985). N. Foote, J. Petersen, P. M. A. Gadsby, C. Greenwood, and A. J. Thomson, Biochem. Z. 230,227 (1985). C. Greenwood, N. Foote, P. M. A. Gadsby, and A. J. Thomson, Chimicu. Sctipta. 28A, 79 (1988). M. Ron&erg, N. Ellfolk, and H. B. Dunford, Acta Chem. Scund. B38,79 (19841. R. Aasa, N. Ellfolk, M. Ronnberg, and T. Vanngard, FEBS Let&. 250,175 (1989). M. Ron&erg, K. Qsterlund, and N. Ellfolk, Biochim. Biophys. Acta 670, 170 (1981). N. ElIfolk, M. Ron&erg, R. Aasa, T. Vanngard, and J. Angstrom, Biochim. Biophys. Acra 791, 9 (1984). M. Ron&erg, A. M. Lambier, N. Ellfolk, and H. B. Dunford, Arch. Biuchem. Bicphys. 236,714 (1985). M. Ronnberg, T. Araison, N. Ellfolk, and H. B. Dunford, J. Biol. Chem 256, 2471 (1981). N. Foote, R. Turner, T. Brittain, and C. Greenwood, Biochem J. (in press). N. Foote, A. J. Thomson, D. Barber, and C. Greenwood, Bbchem J.2@9,701(1983). R. Soininen and N. Ellfoik, Acfa Chem. Scund. 26,861 09721. R. Soininen and N. ElIfolk, Acta Chem. Scat&. 27,35 (19731. Q. H. Gibson and L Mimes, Biochem. J 91,161(1964). S. Strickland, G. Palmer, and V. Massey, Z. Biol. Gem. 250,4048 ( 1975). 0. Farver and I. Pecht, Zsmel. J. Chem:21, 13 (1981). 0. Farver, Y. Blatt, and I. Pecht, Biochemistty 21,3556 (1982). M. van de Ramp, R. Floris, F. C. Hali, and G. W. Canters, J. Amer. Chem. Sot. 112, 907 (1990). C. M. Groenveld and G. W. Canters, Eur. J. Biochem. 153,559 (1985). C. M. Groenveld and G. W. Canters, Z. Biol. Gem. 263,167 (1988). M. van de Ramp, M. C. Silvestrini, M. Brunori, J. V. Beeuman, F. C. Hali, and G. W. Canters, Eur. J. Biochem. 194, 109 (1990). R. Aasa, N. Ellfoik, M. Ron&erg, and T. Vanngard, Biochim. Biophys. Acta 670,170 (1981). N. Ellfolk, M. Ron&erg, and K. Osterlund, Biochim. Biophys. Actu 1080, 68 (1991). M. van de Ramp, F. C. Hali, N. Rosa@ A. Finazzi-Agro, and G. W. Canters, B&him. Biophys. Acta 1019,283 (1990).
Received December 23, 1991; accepted March 3, 1992
