240s Biochemical SocietyTransactions ( 1 992) 20 L i g a n d binding properties of bacterial oxidases i n relation t o cytochrome-c oxidase. A. JOHN MOODY, ROBERT B. GENNIS+, DAVID B. HICKS*, JOHN INGLEDEW**, TERRYANN KRULWICH*, JON c. RUMBLEY and PETER R. RICH
w.
+
Glynn Research Institute, Bodmin, Cornwall PL30 4AU, U.K. + Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, U.S.A. **Department of Biochemistry, University of St. Andrews, St. Andrews KY 16 9AL, U.K. *Department of Biochemistry, Mount Sinai School of Medicine, NY 10029, U.S.A. Cytochrome-c oxidase (aa3) isolated from bovine heart shows well-documented ligand binding phenomena. A s prepared the enzyme may contain two subpopulations: ‘slow’ oxidase, which binds ligands extremely slowly, e.g. k = 0.01-0.02 M-l.s-I f o r cyanide a t 20’C, and ‘fast’ oxidase which binds ligands much more rapidly, e.g. k - 2 M-l.s-I f o r cyanide 111. Both these forms bind ligands much slower than simpler haem-containing enzymes such a s cytchrome-c peroxidase and horseradish peroxidase which have rate constants for cyanide binding greater than lo5 M-l.s-l [2]. However, during turnover ligand binding may be much more rapid, e.g. the rate of onset of inhibition of oxidase by cyanide has a rate constant approaching lo4 M-l.s-l [3]. This ‘closed’/‘open’ phenomenon also applies t o azide but not t o formate where the rate of onset of inhibition ( & - 1 M-j.s-l at p H 7.4, Ref.4) during turnover is comparable t o the rate of binding t o ‘fast’ oxidase (biphasic, k = 0.6 and 0.07 M-l.s-I, respectively, at p H 7.5, Ref. 1). We have made preliminary studies on the ligand binding characteristics of two bacterial terminal oxidases, i.e. cytochrome caaj isolated from the alkalophile B. f i r m u s OF4 and the quinol oxidase cytochrome b o isolated from E . c o l i . These, together with bovine heart cytochrome 003, form part of a family of enzymes which show structural and functional similarity 151. The aim of these studies was t o determine whether the ‘fast’/‘slow’ and ‘closed’/‘open’ phenomena a r e also found in the bacterial oxidases.
caa3 1
With 2 m M KCN cytochrome coo3 appears to hind cyanide monophasically (measured at 432-412 nm) with k = 6 M-l.s-l. Under similar conditions cytochrome b o shows hiphasic hinding kinetics. The two kinetic phases differ in rate by about a n order of magnitude. For the fast phase k = 18 M-l.s-I. When measured at 418-446 nm, t o eliminate contributions from the partial reduction of haem b , the two phases have about the same extent. With higher concentrations of cyanide cytoehrome coo3 shows a second slower phase of cyanide binding (& = 0.02 M-I.s-l with 40 mM KCN, measured at 434-412 nm to eliminate contributions from the partial reduction of haem a ) . The presence of this phase is implied by the low extinction coefficient (approx. 30 mM-l.cm-l) found for AA434-412n after 20 min incubation with 2 mM KCN (compared with 48 mMT.cm-l for ‘fast’ aa3). A similar slow phase is seen in some preparations of cytochrome b o (k = 0.05 M-I.s-I with 20 mM KCN). Hence, it seems that both eytochromcs cao3 and b o have ‘fast’ and ‘slow’ forms with cyanide binding properties like the analogous forms of cytochrome 003. This is further supported by the observation that incubation of cytochrome caa3 at pH 6.5, which with cytochrome a03 causes the ‘fast’ form t o convert to the ‘slow’ form, leads to a decrease in the extent of the fast phase of cyanide binding. W e tentatively equate the intermediate phase of cyanide binding found with cytochrome b o with the chloride-ligated form of the enzyme, again by analogy with cytochrome a03 111. A s well a s showing the ‘fast’/’slow’ phenomenon, both bacterial oxidases show the ‘closed’/‘open’ phenomenon. This is illustrated in Fig. 1 f o r cytochrome cao3, where, with 1 0 p M KCN, maximal inhibition is reached in less than 2 0 0 s consistent with k > lo3 M-l.s-l. A value in excess of lo3 M-I.s-I is also found for cytochrome bo. The formate-binding kinctics shown by cytochrome caa3 at p H 6.5 a r e similar t o those found for ‘fast’cytochrome 003 (biphasic, k = 2 and 0.2 M-l.s-l). The rate of formatc binding t o cytochrome b o is much slower but biphasic kinetics a r e also found ( k f o r the fast phase = 0.01 M-l.s-I at pH 7.2) and the pH-dependence is like that found in cytochrome 003, i.e. an increase in rate a s the p H decreases from 8.2 t o 5.4. Cytochrome b o is different from the other two oxidases in that the formate-binding spectrum (peak 403-404 nm, trough 416-417 o m ) is not the inverse of the cyanide-binding spectrum (peak 420-421 nm, trough 400nm) in the Soret region. There is also a dramatic difference in the effects of H 2 0 2 on bo when compared with aa3and caaj. H 2 0 2 binds to cytochrome b o (in membranes from E. c o l i RG145) to form a single species that appears analogous to the ‘peroxy’ form of aa3 ( k = 800 M-I.s-I and Kd = % I W ~ M at pH 8.0). However, with both aa3 and caaj further reaction of the ‘peroxy’ form occurs t o produce the ‘ferryl’ form 161. In conclusion we find a high degree of similarity hetween the hinuclear centre ligand hinding reactions of bovine heart cytochrome ao3, B. f i r m u s OF4 cytochrome caaj and E. coli cytoehrome bo. The finding that the ‘fast’/‘slow’ and ‘elosed’/‘open’ phenomena also occur in these eniymes suggests they are intrinsic properties of the hinuclear Cu-Fe type of terminal oxidase. This work was supported by the S E R C (grant GR/F/17605). l.Moody, A.J., Cooper, C.E. and Rich, P.R. (1991) Biochim. Biophys. Acta 1059, 189-207 2. Dunford, H.B. and Nadeyhdin, A.D. (1982) Oxidascs and related redox systems (King, T.E., Mason, H.S. and Morrison, M., cds.), pp. 653-660, Pergamon, Oxford 3. Mitchell, R., Brown, S., Mitchell, P. and Rich, P.R. (1902) Biochim. Biophys. Aeta, in press 4. Nicholls, P. (1976) Biochim. Biophys. Acta 430, 13-29 5 . Saraste, M., Holm, L., Lemieux, L., Luhhcn, M. and van der Oost. J . (1991) Biochem. Soc. Trans. 10, 608-612 6. Moody, A.J. (1991) Biochcm. Soc. Trans. 19, 617-022
Fig. 1 & onset
B-V
of
.
. ..
inhibition
of
cvtochrome
coo 3
from
Turnover was started by addition of 15 nM enzyme to 50 mM MES pH 6.0, containing 0.1% Triton X-100, 1 6 p M cyt. c 2 + and KCN a s indicated. The oxidation of the cyt. c 2 + was monitored a s the decrease in A A 5 5 0 - 5 4 0 ~ ~ .
w.
+
Glynn Research Institute, Bodmin, Cornwall PL30 4AU, U.K. + Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, U.S.A. **Department of Biochemistry, University of St. Andrews, St. Andrews KY 16 9AL, U.K. *Department of Biochemistry, Mount Sinai School of Medicine, NY 10029, U.S.A. Cytochrome-c oxidase (aa3) isolated from bovine heart shows well-documented ligand binding phenomena. A s prepared the enzyme may contain two subpopulations: ‘slow’ oxidase, which binds ligands extremely slowly, e.g. k = 0.01-0.02 M-l.s-I f o r cyanide a t 20’C, and ‘fast’ oxidase which binds ligands much more rapidly, e.g. k - 2 M-l.s-I f o r cyanide 111. Both these forms bind ligands much slower than simpler haem-containing enzymes such a s cytchrome-c peroxidase and horseradish peroxidase which have rate constants for cyanide binding greater than lo5 M-l.s-l [2]. However, during turnover ligand binding may be much more rapid, e.g. the rate of onset of inhibition of oxidase by cyanide has a rate constant approaching lo4 M-l.s-l [3]. This ‘closed’/‘open’ phenomenon also applies t o azide but not t o formate where the rate of onset of inhibition ( & - 1 M-j.s-l at p H 7.4, Ref.4) during turnover is comparable t o the rate of binding t o ‘fast’ oxidase (biphasic, k = 0.6 and 0.07 M-l.s-I, respectively, at p H 7.5, Ref. 1). We have made preliminary studies on the ligand binding characteristics of two bacterial terminal oxidases, i.e. cytochrome caaj isolated from the alkalophile B. f i r m u s OF4 and the quinol oxidase cytochrome b o isolated from E . c o l i . These, together with bovine heart cytochrome 003, form part of a family of enzymes which show structural and functional similarity 151. The aim of these studies was t o determine whether the ‘fast’/‘slow’ and ‘closed’/‘open’ phenomena a r e also found in the bacterial oxidases.
caa3 1
With 2 m M KCN cytochrome coo3 appears to hind cyanide monophasically (measured at 432-412 nm) with k = 6 M-l.s-l. Under similar conditions cytochrome b o shows hiphasic hinding kinetics. The two kinetic phases differ in rate by about a n order of magnitude. For the fast phase k = 18 M-l.s-I. When measured at 418-446 nm, t o eliminate contributions from the partial reduction of haem b , the two phases have about the same extent. With higher concentrations of cyanide cytoehrome coo3 shows a second slower phase of cyanide binding (& = 0.02 M-I.s-l with 40 mM KCN, measured at 434-412 nm to eliminate contributions from the partial reduction of haem a ) . The presence of this phase is implied by the low extinction coefficient (approx. 30 mM-l.cm-l) found for AA434-412n after 20 min incubation with 2 mM KCN (compared with 48 mMT.cm-l for ‘fast’ aa3). A similar slow phase is seen in some preparations of cytochrome b o (k = 0.05 M-I.s-I with 20 mM KCN). Hence, it seems that both eytochromcs cao3 and b o have ‘fast’ and ‘slow’ forms with cyanide binding properties like the analogous forms of cytochrome 003. This is further supported by the observation that incubation of cytochrome caa3 at pH 6.5, which with cytochrome a03 causes the ‘fast’ form t o convert to the ‘slow’ form, leads to a decrease in the extent of the fast phase of cyanide binding. W e tentatively equate the intermediate phase of cyanide binding found with cytochrome b o with the chloride-ligated form of the enzyme, again by analogy with cytochrome a03 111. A s well a s showing the ‘fast’/’slow’ phenomenon, both bacterial oxidases show the ‘closed’/‘open’ phenomenon. This is illustrated in Fig. 1 f o r cytochrome cao3, where, with 1 0 p M KCN, maximal inhibition is reached in less than 2 0 0 s consistent with k > lo3 M-l.s-l. A value in excess of lo3 M-I.s-I is also found for cytochrome bo. The formate-binding kinctics shown by cytochrome caa3 at p H 6.5 a r e similar t o those found for ‘fast’cytochrome 003 (biphasic, k = 2 and 0.2 M-l.s-l). The rate of formatc binding t o cytochrome b o is much slower but biphasic kinetics a r e also found ( k f o r the fast phase = 0.01 M-l.s-I at pH 7.2) and the pH-dependence is like that found in cytochrome 003, i.e. an increase in rate a s the p H decreases from 8.2 t o 5.4. Cytochrome b o is different from the other two oxidases in that the formate-binding spectrum (peak 403-404 nm, trough 416-417 o m ) is not the inverse of the cyanide-binding spectrum (peak 420-421 nm, trough 400nm) in the Soret region. There is also a dramatic difference in the effects of H 2 0 2 on bo when compared with aa3and caaj. H 2 0 2 binds to cytochrome b o (in membranes from E. c o l i RG145) to form a single species that appears analogous to the ‘peroxy’ form of aa3 ( k = 800 M-I.s-I and Kd = % I W ~ M at pH 8.0). However, with both aa3 and caaj further reaction of the ‘peroxy’ form occurs t o produce the ‘ferryl’ form 161. In conclusion we find a high degree of similarity hetween the hinuclear centre ligand hinding reactions of bovine heart cytochrome ao3, B. f i r m u s OF4 cytochrome caaj and E. coli cytoehrome bo. The finding that the ‘fast’/‘slow’ and ‘elosed’/‘open’ phenomena also occur in these eniymes suggests they are intrinsic properties of the hinuclear Cu-Fe type of terminal oxidase. This work was supported by the S E R C (grant GR/F/17605). l.Moody, A.J., Cooper, C.E. and Rich, P.R. (1991) Biochim. Biophys. Acta 1059, 189-207 2. Dunford, H.B. and Nadeyhdin, A.D. (1982) Oxidascs and related redox systems (King, T.E., Mason, H.S. and Morrison, M., cds.), pp. 653-660, Pergamon, Oxford 3. Mitchell, R., Brown, S., Mitchell, P. and Rich, P.R. (1902) Biochim. Biophys. Aeta, in press 4. Nicholls, P. (1976) Biochim. Biophys. Acta 430, 13-29 5 . Saraste, M., Holm, L., Lemieux, L., Luhhcn, M. and van der Oost. J . (1991) Biochem. Soc. Trans. 10, 608-612 6. Moody, A.J. (1991) Biochcm. Soc. Trans. 19, 617-022
Fig. 1 & onset
B-V
of
.
. ..
inhibition
of
cvtochrome
coo 3
from
Turnover was started by addition of 15 nM enzyme to 50 mM MES pH 6.0, containing 0.1% Triton X-100, 1 6 p M cyt. c 2 + and KCN a s indicated. The oxidation of the cyt. c 2 + was monitored a s the decrease in A A 5 5 0 - 5 4 0 ~ ~ .
