AbstractA common feature within the heme-copper oxidase superfamily is the dinuclear heme-copper center. Analysis via extended X-ray absorption fine structure (EXAFS) has led to the proposal that sulfur may be bound to CUB, a component of the dinuclear center, and a highly conserved methionine (M110 in the E. coli oxidase) in subunit I has been proposed as the ligand. Recent models of subunit I, however, suggest that this residue is unlikely to be near CUB, but is predicted to be near the low spin heme component of the heme-copper oxidases. In this paper, the role of M110 is examined by spectroscopic analyses of site-directed mutants of the bo3-type oxidase from Escherichia coli. The results show that M110 is a non-essential residue and suggest that it is probably not near the heme-copper dinuclear center

Alben, James O.

Calhoun, Melissa W.

Garcia-Horsman, J. Arturo

Gennis, Robert B.

Lemieux, Laura J.

Thomas, Jeffrey W.

Elsevier - Publisher Connector 

FEBS 15768 FEBS Letters 368 (1995) 523-525 
The highly conserved methionine of subunit I of the heme-copper oxidases 
is not at the heme-copper dinuclear center: mutagenesis of M110 in subunit 
I of cytochrome bo3-type ubiquinol oxidase from Escherichia coli 
Melissa W. Calhoun a, Laura J. Lemieux a'**, J. Arturo Garcia-Horsman a'***, Jeffrey W. Thomas a, 
James O. Alben b, Robert  B. Gennis a'* 
~School f Chemical Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA 
bDepartment of Medical Biochemistry, Ohio State University, Columbus, OH 43210, USA 
Received 10 April 1995; revised version received 7June 1995 
Abstract A common feature within the heine-copper oxidase 
superfamily is the dinuclear heine-copper center. Analysis via 
extended X-ray absorption fine structure (EXAFS) has led to the 
proposal that sulfur may be bound to CUB, a component of the 
dinuclear center, and a highly conserved methionine (Ml l0  in the 
E. coil oxidase) in snbanit I has been proposed as the ligand. 
Recent models of subunit I, however, suggest hat this residue is 
unlikely to be near CUB, but is predicted to be near the low spin 
heine component of the heine-copper oxidases. In this paper, the 
role of M l l0  is examined by spectroscopic analyses of site- 
directed mutants of the bo~-type oxidase from Escherichia coil 
The results show that M l l0  is a non-essential residue and 
suggest hat it is probably not near the heine-copper dinuclear 
center. 
Key words." ???? 
subunit I of the heme-copper oxidases. This methionine, which 
is present in all but three species, has been proposed as a ligand 
to Cu B in the binuclear center [5]. However, this is incompatible 
with recent models of subunit I based on site-directed mut- 
agenesis tudies of two bacterial heme-copper oxidases, the 
bo3-type oxidase from E. coli and aa3-type oxidase from Rho- 
dobacter sphaeroides [4,7]. In these models, MI I0 is located 
four residues below the histidine (H106) identified as a ligand 
for the low spin heine b component of the E. coli oxidase [8,9], 
which corresponds to the heme a component of the aa3-type 
oxidases [4,7,10,11]. In this work, the role of Ml l0  in the 
bo3-type oxidase from E. coli was examined by site-directed 
mutagenesis. The results clearly show that M l l0  is not an 
essential residue and is unlikely to be near the dinuclear center, 
consistent with the current models of subunit I of the heme- 
copper oxidases [4,7,10]. 
1. Introduction 
The origin of the magnetic coupling in the heme-copper dinu- 
clear center of cytochrome c oxidase has been a subject of 
controversy for over 20 years. Several bridging ligands, both 
endogenous and exogenous, have been proposed to bridge be- 
tween the iron atom of heine a 3 and CuB. Candidates have 
include histidine, sulfide, chloride, ¢t-oxo, tyrosinate, and car- 
boxylate bridges [1]. A sulfur or chloride ligand has been sug- 
gested based upon analysis of EXAFS data [2,3]. A sulfur atom 
has also been proposed in the bo3-type oxidase, another mem- 
ber of the heme-copper oxidase superfamily [4], based upon 
curve-fitting of EXAFS data [5]. 
Sequence alignments of heme-copper oxidases from over 75 
species reveal that subunit I, which binds the dinuclear center, 
contains no invariant cysteine residues [6]. One highly con- 
served methionine (Ml l0 in the E. eoli oxidase) is found in 
*Corresponding author. Fax: (1) (217) 244~3186. 
E-mail: gennis@aries.scs.niuc.edu 
**Current address: Department ofPathology, Richardson Laboratory, 
Queen's University, Kingston, Ontario K7L3N6, Canada. 
***Current address: Department ofMicrobiology, Institute of Cellular 
Physiology, National Autonomous University of Mexico, Mexico, 
DFCP 04510. 
Abbreviations: EPR, electron paramagnetic resonance spectroscopy; 
EXAFS, extended X-ray fine structure spectroscopy; FTIR, Fourier 
transform infrared spectroscopy. 
2. Materials and methods 
Mutants were constructed and confirmed by DNA sequencing ac- 
cording to previously punished methods [8]. To test enzyme function, 
complementation analysis was performed in strain GO105 and strain 
RG129, as previously outlined [8]. These strains contain mutations in 
the cyo gene and the cyd gene and are recombination-defective (recA) 
[8,12] Expression of the mutants were performed in strain GL 101 (cyo 
sdh recA) or in strain GO105 (cyo Acyd recA). 
Under anaerobic onditions, expression of the bo3-type oxidase is 
greatly repressed [13,14]. To grow E. coli aerobically on non-fermenta- 
ble substrates, a functional terminal oxidase must be present [15]. Ex- 
pression of mutants which grow poorly under aerobic onditions re- 
quires a functional form of the bd-type oxidase, which has three heme 
components, b558, b595, and d [16 18]. Two mutants were examined in
this study. Mll0A is fully functional, and was examined in host 
strain GO105 with no other oxidase present. Mll0I, however, re- 
sulted in slow growth of GO105, and therefore this mutant was exam- 
ined in host strain GL101, which contains the alternate bd-type quinol 
oxidase. 
EPR and visible spectroscopies were performed with membrane 
preparations as previously described [8]. Activity assays were per- 
formed spectrophotometrically by measuring the oxidation of NADH 
[19]. Cryogenic Fourier transform infrared absorption difference spec- 
troscopy of CO adducts of the E. coli oxidase was performed as previ- 
ously described [19-21]. 
3. Results 
Two mutants were constructed for this study, in which M110 
is substituted by alanine (M 110A) and isoleucine (M 110I). Both 
the M110I and M 110A mutants complement oxidase-deficient 
strains RG129 and GO105, demonstrating that both mutant 
0014-5793/95/$9.50 © 1995 Federation of European Biochemical Societies. All rights reserved. 
SSDI 0014-5793(95)00696-6 
524 M. W. Calhoun et aL IFEBS Letters 368 (1995) 523-525 
500 550 600 650 700 
Wavelength (nm) 
Fig. 1. The ~- and fl-bands of wildtype cytochrome bo 3 and M110 
mutant oxidases. Samples contain 8 mg membrane protein/mL. Spectra 
were recorded at 77 k and are presented as dithionite-reduced minus 
air-oxidized visible spectra. The split ~-band at 556 nm and 564 nm is 
characteristic of the wildtype enzyme. This split is nearly absent from 
the M110I mutant, but this is somewhat variable in different prepara- 
tions. The top three spectra contain a minor amount of the cytochrome 
bd quinol oxidase from the background strain GL101. The bottom two 
spectra are inbackground strain GO105. 
enzymes are functional in vivo. However, the M110I mutant 
grows more slowly than the wildtype control on minimal agar 
plates as well as on rich media supplemented with lactate. 
Activity assays how that the specific activity of M110I is 45% 
of the wildtype enzyme. 
Visible dithionite-reduced minus air-oxidized spectra of the 
membranes containing the wildtype and mutant oxidases are 
shown in Fig. 1. In each case, the intensity of the absorbance 
clearly indicates that the oxidase is overproduced. The low spin 
heine b component of the wildtype enzyme has a split c~-band 
with features near 556 nm and 564 nm. The spectrum of M110A 
is very similar to that of the wildtype oxidase, with perhaps a
slightly broadened split s-band. The spectroscopic properties 
of M1101 vary somewhat in independent membrane prepara- 
tions. In most membrane preparations, M110I has a relatively 
diminished absorbance at 564 nm in the difference spectrum, 
and the split c~-band is much less pronounced. However, in 
some preparations of M110I, the split s-band is much more 
apparent. 
The EPR spectrum of wildtype cytochrome bo~ contains 
features ofheme b, which is low spin, with g values at 2.98, 2.22, 
and 1.47. EPR spectroscopy of Ml l0 I  reve~led: small changes 
in the low spin heme signal (not shown), with the g"z value at 
3.00, while the gy remains at 2.22. 
The EPR spectrum of the wildtype enzyme also contains a
small signal at g = 6 from the high spin heme o3 [22]. Magnetic 
interaction of CuB with heme o3 results in a quenched signal at 
g = 6, and the intensity of this attenuated signal corresponds 
to much less than one heine per enzyme molecule. This spin 
coupling also renders Cug + EPR silent. If the sulfur of M110 
formed a bridge between heme 03 and CuB, the loss of this 
bridging atom might be expected to remove the spin coupling. 
The result should be a large increase in the high spin signal at 
g = 6, as well as the appearance of an EPR signal from the 
copper. However, the amount of the g = 6 signal in the EPR 
spectrum of M110I is comparable to that in the wildtype (not 
shown), demonstrating that the spin coupling still exists. No 
copper EPR signals are present near g = 2 in the EPR spectrum 
of M110I. By this criterion, the heine-copper binuclear center 
is unperturbed by the M110A and M110I mutations. 
Another probe of the integrity of the dinuclear center is the 
FTIR spectrum of CO bound to the oxidase. CO forms a stable 
complex with heme o3 under ambient conditions. Upon photol- 
ysis of the iron-carbon bond, CO is free to interact with CuB 
[23], and will form a stable CuB-CO adduct at cryogenic tem- 
peratures [23,24]. The CO adduct of the M 110I mutant exhibits 
an FTIR difference spectrum which is indistinguishable from 
that of the wildtype oxidase (Fig. 2). 
4. Discussion 
Both genetic omplementation and the in vitro quinol oxi- 
dase assays demonstrate that M l l0  can be substituted by 
0_006 
0.001 ~ 
A b -0.004 
8 
o 
r 
b 
n -0.009 
c 
e 
-0.014 
-o.o19 . . . .  ', . . . .  ~ . . . .  I . . . .  I . . . .  I . . . .  I . . . .  I . . . .  
1900 1925 1950 1975 2000 2025 2050 2075 2100 
Wavenumber (cm-1) 
Fig. 2. Low temperature FTIR difference spectrum of the CO adduct 
of the M110I mutant oxidase, recorded using a membrane preparation 
containing the mutant. The difference spectrum is identical to that of 
the wildtype, with the Fe-CO band at 1959 cm -~ and the CuB-CO 
envelope near 2063 cm -1. The Fe-CO band at 1984 cm -l arises from 
cytochrome bd, which is also present i  the membranes [26]. The spikes 
in the spectrum are water vapor. 
M.W. Calhoun et al./FEBS Letters 368 (1995) 523 525 
another residue without elimination of enzymatic activity. 
M 110A i~ indistinguishable from the wildtype oxidase, whereas 
M l l0 I  has about 45% of the specific activity of the wildtype 
control. Therefore, although this menthionine is very highly 
conserved among the heme-copper oxidases, it is not essential 
for the activity of the bo3-type quinol oxidase. 
In the current model of the heme-copper oxidases, M110 is 
one e-helical turn below H106, placing the sulfur of the 
methionine side chain near the periphery of the low spin heine 
b [4,7]. This model is incompatible with the suggestion that the 
sulfur atom of M110 is within the ligation sphere of the high 
spin heme o 3 or Cu B [5]. The data presented definitively demon- 
strate that the substitutions for M110 do not perturb the integ- 
rity of the heme-copper dinuclear center. Hence, the sulfur 
atom of M110 is very unlikely to be a bridging ligand in cyto- 
chrome bo3. By analogy, the equivalent methionines in the other 
members of the heine-copper superfamily are also unlikely to 
be located at the dinuclear center of these enzymes. 
Whereas the M110A mutant has virtually identical enzymatic 
and spectroscopic properties as the wildtype, the isoleucine 
substitution at this position (M110I) has subtle affects on the 
visible and EPR spectra of the low spin heme b component of 
the oxidase. These changes may be due in part to the substitu- 
tion of heine O for heme B at the low spin heme site, resulting 
in an oo3-type oxidase, as has been previously reported [25]. 
Heme O causes narrowing and a blue-shift of the c~-band, ap- 
parently due to the restoration of the symmetry of the heine 
electronic transitions [25]. This explanation would also be con- 
sistent with the variability of the spectroscopic properties of 
M 110I, since this phenomenon is sensitive to growth conditions 
[25]. It should be noted that the perturbation caused by M110I 
cannot be simply ascribed to the substitution of heme O at the 
low spin site, since this substitution does not explain the re- 
duced specific activity of this mutant [25]. A more detailed 
analysis would require the purification and full characterization 
of the M 110I oxidase, which is beyond the scope of the present 
work. 
In conclusion, the r sults presented here show that M110 is 
not required for the quinol oxidase activity of cytochrome bo3 
and does not appear to be in the immediate vicinity of the 
dinuclear center. 
Acknowledgements'." This work was supported by grants from the U.S. 
Department ofEnergy (R.B.G.) (DEFG02 87ER13716) and from the 
National Science Foundation (J.O.A.) (DMB894)4614). 
References 
[1] Wikstr6m, M., Krab, K. and Saraste, M. (1981) in: Cytochrome 
Oxidase - a Synthesis, Academic Press, New York, NY. 
525 
[2] Scott, R.A., Li, RM. and Chan, S.I. (1988) Ann. N.Y. Acad. Sci. 
550, 53-58. 
[3] Powers, L., Lauraeus, M., Reddy, K.S., Chance, B. and Wikstr6m, 
M. (1994) Biochim. Biophys. Acta 1183, 504-512. 
[4] Calhoun, M.W., Thomas, J.W. and Gennis, R.B. (1994) TIBS 19, 
325-330. 
[5] Ingledew, W.J. and Bacon, M. (1991) Biochem. Soc. Trans. 19, 
613-616. 
[6] Saraste, M. (1990) Q. Rev. Biophys. 23, 331-366. 
[7] Hosler, J.R, Ferguson-Miller, S., Calhoun, M.W., Thomas, J.W., 
Hill, J., Lemieux, L., Ma, J., Georgiou, C., Fetter, J., Shapleigh, J., 
Tecklenburg, M.M.J., Babcock, G.T. and Gennis, R.B. (1993) 
J. Bioenergetics Biomembranes 25, 121 136. 
[8] Lemieux, L.J., Calhoun, M.W., Thomas, J.W., Ingledew, W.J. and 
Gennis, R.B. (1992) J. Biol. Chem. 267, 2105 2113. 
[9] Minagawa, J., Mogi, T., Gennis, R.B. and Anraku, Y. (1992) 
J. Biol. Chem. 267, 2096~2104. 
[10] Garcia-Horsman, J.A., Barquera, B., Rumbley, J., Ma, J. and 
Gennis, R.B. (1994) J. Bacteriol. 176, 5587-5600. 
[11] Shapleigh, J.E, Hosler, J.E, Tecklenburg, M.M.J., Kim, Y., 
Babcock, G.T., Gennis, R.B. and Ferguson-Miller, S. (1992) Proc. 
Natl. Acad. Sci. USA 89, 47864790. 
[12] Au, D.C.-T. and Gennis, R.B. (1987) J. Bacteriol. 169, 3237- 
3242. 
[13] Iuchi, S., Chepuri, V., Fu, H.-A., Gennis, R.B. and Lin, E.C.C. 
(1990) J. Bacteriol. 172, 6020-6025. 
[14] Minagawa, J., Nakamura, H., Yamato, I., Mogi, T. and Anraku, 
Y. (1991) J. Biol. Chem. 265, Ii198-11203. 
[15] Au, D.C.-T, Lorence, R.M. and Gennis, R.B. (1985)J. Bact. 161, 
123-127. 
[16] Lorence, R.M., Koland, J.G. and Gennis, R.B. (1986) Biochemis- 
try 25, 2314-2321. 
[17] Anraku, Y. and Gennis, R.B. (1987) TIBS 12, 262-266. 
[18] Anraku, Y. (1988) Annu. Rev. Biochem. 57, 101 132. 
[19] Calhoun, M.W., Thomas, J.W., Hill, J.J., Hosler, J.R, Shapleigh, 
J.P., Tecklenburg, M.M.J., Ferguson-Miller, S., Babcock, G.T., 
Alben, J.O. and Gennis, R.B. (1993) Biochemistry 32, 10905- 
10911. 
[20] Hill, J., Goswitz, V.C., Calhoun, M., Garcia-Horsman, J.A., 
Lemieux, L., Alben, J.O. and Gennis, R.B. (1992) Biochemistry 31, 
11435-11440. 
[21] Calhoun, M.W., Lemieux, L.J., Thomas, J.W., Hill, J.J., Goswitz, 
V.C., Alben, J.O. and Gennis, R.B. (1993)Biochemistry 32, 13254- 
13261. 
[22] Salerno, J.C., Bolgiano, B., Poole, R.K., Gennis, R.B. and 
Ingledew, W.J. (1990) J. Biol. Chem. 265,4364-4368. 
[23] Alben, J.O., Moh, EP., Fiamingo, F.G. and Altschuld, R.A. 
(1981) Proc. Natl. Acad. Sci. USA 78, 234-237. 
[24] Dyer, R.B., Einarsd6ttir, O., Killough, EM., L6pez-Garriga, J.J. 
and Woodruff, W.H. (1989) J. Am. Chem. Soc. 1989 111, 7651 
7659. 
[25] Puustinen, A., Morgan, J.E., Verkhovsky, M., Thomas, J.W., 
Gennis, R.B. and Wikstr6m, M. (1992) Biochemistry 31, 10363- 
10369. 
[26] Hill, JJ., Alben, J.O. and Gennis, R.B. (1993) Proc. Natl. Acad. 
Sci. USA 90, 5863 5867. 


The highly conserved methionine of subunit I of the heme-copper oxidases is not at the heme-copper dinuclear center: Mutagenesis of M110 in subunit I of cytochrome bo3-type ubiquinol oxidase from Escherichia coli 

file:///data/core-remote/dit/data/elsevier/pdf/348/aHR0cDovL2FwaS5lbHNldmllci5jb20vY29udGVudC9hcnRpY2xlL3BpaS8wMDE0NTc5Mzk1MDA2OTY3.pdf

The highly conserved methionine of subunit I of the heme-copper oxidases is not at the heme-copper dinuclear center: Mutagenesis of M110 in subunit I of cytochrome bo3-type ubiquinol oxidase from Escherichia coli

Abstract

Similar works

Full text

Available Versions

Elsevier - Publisher Connector

Elsevier - Publisher Connector