MUNICIPALIDADES PROVINCIALES DE LA REGIÓN SAN MARTIN TARAPOTO

All protein determinations were performed according to Lowry et al., 1951 with the inclusion of 1% sodium dodecyl sulphate. All reactions were conducted at 3TC, using bovine serum albumin as a standard. The concentration of cytochromes bo3 and bd was determined using the absorbtion coefficients E5 6 0-5 8O of 18.7 and 14.8mM-icm-i respectively for the low spin b haem from the reduced minus oxidised optical difference spectrum (Kita et al,

1984a,b)

2.22 Materials

E. coli strain BL21(DE3)/pET.E2 was a gift of Dr. M. Saraste, EMBL, Heidelberg, Germany. DH5a and NM522 were obtained

from Pharmacia. All other strains and cyo A mutant W136A were a

kind gift of Prof. R.B. Gennis, University of Illinois at Urbana- Champaign, Illinois, USA.

Decylubiquinone, ubiquinone-1, vitamin Ki and menadione were obtained from Sigma as were octylgluoside, triton X-100 and dodecylmaltoside, HOQNO, DPPH, deuterium oxide and isopropan(oL(Q. Yeast extract, tryptone, and acid-hydrolysed casein were obtained from Merck. Aurachin D and tridecylstigmatellin were a kind gift of Dr. P. Rich, formerly of Glynn Research Institute, Bodmin, Cornwall, UK.

USE mutagenesis kits and Phosphorylation kits were obtained from Pharmacia Biotech. Restriction enzymes and other molecular

biology reagents were obtained form New England Biolabs, USA. All other laboratory reagents including redox dye mediators were obtained form Sigma or Aldrich Chemical Company.

Semiquinone Stabilisation by Cytochrome bos

3.0 SEMIQUINONE STABILISATION

BY CYTOCHROME bos

3.1 In trod u ction

Chapter 1.9 reviewed the recent literature concerning the elucidation of the structure and functional relationships in cytochrome bo^ and chapter 1 . 1 0 focused on the current understanding of quinone binding to the complex. The principle aim of this chapter is to describe experiments designed to probe further the thermodynamics of quinol oxidation and to provide conclusive evidence on the nature of quinone binding and the resultant implications for the catalytic mechanism of the enzyme.

Ingledew et al., 1995, in a study using ESR spectroscopy

described the presence of a ubisemiquinone radical intermediate

associated with the cytochrome bo^ complex during potentiometric

titrations and thermodynamic behaviour of the species. The results were deemed consistent with there being one quinone binding site and that being the site of quinol oxidation. In addition the ESR spectrum of the semiquinone had partially resolved hyperfine splittings at 0.4mT which were unassigned.

Sato-Watanabe et al., 1994b observed that when the

cytochrome bos complex was extracted using dodecylmaltoside, a

stoichiometric amount of ubiquinone- 8 co-purified with the

complex. Extraction with octylglucoside and triton X-100 resulted in a preparation with little or no bound quinone. Based on inhibitor binding studies the quinone molecule was proposed to bind in a second high-affmity site separate from the ubiquinol oxidation site. A further ESR study of this bound quinone gave a semiquinone

species similar to that described by Ingledew et al., 1995 (the

Semiquinone Stabilisation by Cytochrome bo3

1 0 0 extraction and reconstituted with excess ubiquinone analogue) in terms of the thermodynamic properties and the lineshape of the ESR spectrum of the semiquinone (Sato-Watanabe et al., 1995). They concluded that there would appear to be no ESR active

semiquinone at the site of ubiquinol oxidation under the conditions of the potentiometric titrations conducted in both studies.

The aims of the present chapter are five-fold. The first was to reconcile the findings of Ingledew et al., and Sato-Watanabe et al.,

in the light of the ambiguity in quinone binding due to the choice of extraction detergent. To this end potentiometric titrations of the

membrane-bound cytochrome bo^ complex with endogenous

ubiquinone- 8 and of the octyl glucoside/triton X-100 and

dodecylmaltoside preparations with and without excess quinone

I were conducted.

The second was to probe the structure of the quinone binding site giving rise to the ESR spectrum using ubiquinone and menaquinone analogues and other substituted benzoquinones and assessing the effect on the lineshape of the ESR spectrum of the

semiquinone. Deuterium exchange and labelling studies were

conducted in an attempt to identify the species giving rise to the hyperfine splittings of the semiquinone ESR spectrum.

The third was to assess the effect of the inhibitors HOQNO and

tridecylstigmatellin on semiquinone formation during

potentiometric titrations. Both inhibitors are known to act at sites of

quinone binding and inhibit ubiquinol oxidation (Kita et a l, 1984a;

Meunier etal., 1995).

The fourth aim, in view of the availability of a crystal

structure of the cyoA fragment (Wilmanns et aL, 1995) and given

that subunit II has been implicated in quinone binding to the complex (Welter et al., 1994) was to assess this hydrophilic portion of subunit II , using ESR, for the presence of any quinone binding and subsequent semiquinone stabilisation ability.

Finally, the semiquinone radicals of two subunit II mutants,

K II8L and WI36A were studied using ESR. Despite the sequences

of many quinone binding proteins being known including the crystal structures of two there is no overall structural motif to identify an amino acid residue as being part of a quinone binding site. The sequences of all the known haem-copper quinol oxidases were

aligned with three cytochrome c oxidases. Differences between the

two sub-groups were probed by site-directed mutagenesis and the

resultant mutant, K II8L together with WI36A were studied using

Semiquinone Stabilisation by Cytochrome bos