Fast Cytochrome bo from Escherichia coli reacts with azide and nitric ...

1 downloads 0 Views 122KB Size Report
3928 Biochemical Society Transactions (1997) 25. 19 Fast Cytochrome bo from Escherichiu coli reacts with azide and nitric oxide to form a complex analogous ...
3928 Biochemical Society Transactions (1997) 25 19 Fast Cytochrome bo from Escherichiu coli reacts with

azide and nitric oxide to form a complex analogous to that f o m d by cytochrome c oxidase CLIVB S. B L I T L E d ,' MYLES R CHElEMAN*, COLJN GREENWCKd, ANDREW J. THOMSOId and NICHOLASJ. WATMOUGI$

Centre for Metalloprotein Spectroscopy and Biology, %chool of Biological Sciences and hchool of Chemical Sciences, University of East Anglia, Norfolk, NR4 7TJ, U.K. Cytochrome bo from Escherichin coli is a member of the conserved superfamily of haem-copper terminal oxidases which includes mitochondrial cytochrome c oxidase (CCO). In addition to catalysing the four electron reduction of dioxygen to water, these oxidases couple thii reaction to vectorial proton translocation across the membranes in which they are situated. Cytochrome bo is composed of four subunits, three of which are highly conserved with CCO. Subunit I contains all the redox centres; a low-spin haem b, a high-spin haem o and a copper atom (CU,). Recent structural studies of the bovine mitochondria1CCO [l] and Paracoccus denitnj?cans CCO [2] are consistent with earlier mutagenesis and spectroscopic [3] studies of cytochrome bo. These studies have demonstrated that haem b is a magnetically isolated low-spin ferric species and haem o is a histidine-coordinated high-spin ferric species. CUBis ligated by three histidine residues and is magnetically coupled to haem 0, forming the binuclear centre, the site of dioxygen reduction and the presumed site of proton pumping. The purification and characterisation of fast cytodvome bo from this laboratory has been described in detail [3-71. The fast form of the enzyme is defined by characteristic electronic absorption bands at 406.5 and 624 nm, and binds low concentrations (0.05 - 1.2 mM) of cyanide rapidly. Despite the apparent similarity between the binuclear centres of fist cytochmme bo andfast CCO, there are distinct differences in their EPR spectra. Fast cytochrome bo shows unusual broad EPR signals with prominent features at g=7.3 and 3.7 which are thought to arise from a weak magnetic coupling between high-spin haem o (S=5/2) and Cugu (S=1/2), whereas fast CCO does not. The binding of the exogenous ligand azide to CuB of cytochrome bo [qcauses a shift in these broad signals, but retains the pattern of one broad derivative below loOmT and second feature in the 150-25OmT region. The azide bound form of CCO is EPR silent Furthermore, CCO forms a complex in the presence of azide and nitric oxide (NO) which exhibits EPR broad features downfield from g=2 and a sharp triplet near g=4 [8]. This has been attributed to a triplet state formed by the interaction of aJI-NO (S=1/2)and Cu# (S=1/2), with tlie triplet near g=4 due to AM, = 2 transitions. It has been reported that purified cytochrome bo is unable to form this complex [Q], however there is some evidence for the formation of a azide-NO complex using membrane preparations of cytochrome bo [lo]. In order to resolve this discrepancy we have reexamined the reaction between azide boundcytochrome bo and NO. Figure 1 shows the EPR spectrum of the resultant complex which is similar to that of the corresponding CCO complex. The spectrum shows the characteristic triplet species with signals near g=2 and ~ 4 . 4 .The sharp feature centred at g=4.4 exhibits the typical four-line hyperfine pattern with a splitting of 9.3 mT due to the copper nucleus, consistent with' the 9.7 mT reported for CCO [S). The formation of the same complex with azide and NO

R3mT

L

1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0

MagneticField (mT)

Figure 1. X-band EPR spednnn of the cytochnrme bo azide NO complex. Conditionsofmeasureme&: Tsmperpture lOK, Miorowavepower2.03mW,Modulationamplitude1mT demonstrates the similarity of the binuclear centres of cytochrome bo and CCO. We therefore suggest that the EPR broad signals exhibited byfast and azide-bound cytochrome bo may reflect only subtle differences in the binuclear centres of these oxidases. This work was supported by grants from BBSRC (CG, AJT)

and the Wellcome Trust (NJW). 1. Tsukihara, T., Aoyama, H.,Yamashita, E., Tomizaki, T., Yamaguchi, H.,Shinzawa-Itoh., K., Nakashima, R., Yaono, R. and Yoshikawa, S. (1995) Science 269,1069-

1074 2. Iwata, S.,Ostermeier, C., Ludwig, B. and Michel, H. (1995) Nature 376,660-669 3. Cheesman, M.R., Watmough, N.J., Pires, C. A., Turner, R., Brittain, T., Gennis, R. B., Greenwood, C. and Thomson, A. J. (1993) Biochem. J. 289,709-718 4. Cheesman, M. R., Watmough, N. J., Gennis, R. B., Greenwood, C. and Thomson, A. J. (1994) Eur. J. Biochem. 219,595-602 5. Watmough, N. J., Cheesman, M. R., Gennis, R. B., Greenwood, C. and Thomson, A. J. (1993) FEBS Lett. 319, 151-154 6. Watmough, N. J., Cheesman, M. R., Greenwood, C. and Thomson, A. J. (1994)Biochem. J.300,469-475 7. Little, R. H.,Cheesman, M. R., Thomson, A. J., Greenwood, C. and Watmough, N. I. (1996) Biochemistry 35,13780-13787 8. Stevens, T, H., Brudvig, G. W., Bocian, D. F. and Chan, S. I. (1979) Proc. Natl. Acad. Sci. USA. 76,3320-3324 9. Tsubaki, M.,Mogi, T., Anraku, Y. and Hori, H.(1993) Biochemistry 32,6065-6072 10. Cahoun, U W.,Cennis, R. B., Ingledew, W. J. and Salemo, J. C. (1994)Biochim. Biophys. Acta 1206,143-154