Probing the ubiquinol-binding site of recombinant Sauromatum guttatum alternative oxidase expressed in E. coli membranes through site-directed mutagenesis

Abstract

In the present paper we have investigated the effect of mutagenesis of a number of highly conserved residues (R159, D163, L177 and L267) which we have recently shown to line the hydrophobic inhibitor/substrate cavity in the alternative oxidases (AOXs). Measurements of respiratory activity in rSgAOX expressed in Escherichia coli FN102 membranes indicate that all mutants result in a decrease in maximum activity of AOX and in some cases (D163 and L177) a decrease in the apparent Km (O2). Of particular importance was the finding that when the L177 and L267 residues, which appear to cause a bottleneck in the hydrophobic cavity, are mutated to alanine the sensitivity to AOX antagonists is reduced. When non-AOX anti-malarial inhibitors were also tested against these mutants widening the bottleneck through removal of isobutyl side chain allowed access of these bulkier inhibitors to the active-site and resulted in inhibition. Results are discussed in terms of how these mutations have altered the way in which the AOX's catalytic cycle is controlled and since maximum activity is decreased we predict that such mutations result in an increase in the steady state level of at least one O2-derived AOX intermediate. Such mutations should therefore prove to be useful in future stopped-flow and electron paramagnetic resonance experiments in attempts to understand the catalytic cycle of the alternative oxidase which may prove to be important in future rational drug design to treat diseases such as trypanosomiasis. Furthermore since single amino acid mutations in inhibitor/substrate pockets have been found to be the cause of multi-drug resistant strains of malaria, the decrease in sensitivity to main AOX antagonists observed in the L-mutants studied in this report suggests that an emergence of drug resistance to trypanosomiasis may also be possible. Therefore we suggest that the design of future AOX inhibitors should have structures that are less reliant on the orientation by the two-leucine residues. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

Correlating kinetic and structural data on ubiquinone binding and reduction by respiratory complex I

“…To use the NADH:O 2 reaction to investigate ubiquinone reduction by complex I it must be rate limiting. Thus, the NADH (200 μM) and O 2 (200-250 μM) concentrations used are substantially higher than the K M values of 79 ± 8 μM for complex I (measured with the NADH:O 2 reaction) and 10-20 μM for AOX (24). Furthermore, the k cat value for NADH oxidation by the flavin in bovine complex I is >5,000 s −1 (25), more than 10 times faster than the maximum rate of NADH:ubiquinone oxidoreduction, so NADH oxidation does not limit catalysis.…”

Considerations of AOX Functionality Revealed by Critical Motifs and Unique Domains

“…Mutation of any one of these six residues is extremely detrimental to AOX activity. In addition, research has shown that specific mutations in other critical residues partially or completely inactivate AOX [ 24 , 25 , 26 , 27 , 28 , 29 ]. Overall, these important residues are conserved between TbAOX, wheat AOX, HvAOX, and OsAOX isoforms, underscoring their relevance across species ( Table S2 ), [ 12 ].…”

“…The conserved T219S substitution seen in the monocots above may have steric implications, due to the lost methyl group, and this may affect the three-dimensional structure of the protein and change functionality in ways that are currently unknown. There is also an R96A substitution in OsAOX1d which, in other species, causes a drastic loss of activity ( Figure 2 , Table S2 ) [ 28 ]. A deterioration of OsAOX1d efficiency is, therefore, a viable hypothesis.…”

“…It may be helpful to first mutate the residues known to be conserved between all the monocots and TbAOX. Out of these nine, there are mutation studies on three of them (L122, Y198, L212) all of which have shown over 50% loss of activity [ 24 , 28 , 29 ]. There is, therefore, an opportunity to investigate the other six conserved residues.…”

“…The T. brucei AOX residues are in bold. * Indicates residues from Young et al [ 28 ] and Crichton et al [ 29 ]. ** Indicates residues Shiba et al [ 27 ].…”