A structural model of the alternative oxidase of plant mitochondria.

Abstract

The alternative oxidase branches from the main mitochondrial respiratory chain at the level of the ubiquinone pool, it i s non-protonmotive and an integral membrane protein with its active site located on the matrix side of the inner membrane [ I]. Activity of the alternative oxidase strongly correlates with the appearance of three proteins with Mr of 35, 36 and 37 k D in thermogenic tissues and 35 kD in non-thermogenic tissues [2]. Monoclonal antibodies cross-react not only with proteins from aroid (thermogenic) species but also with proteins from widely divergent plant species such as tobacco, pea and potato in addition to fungi and trypanosomes suggesting that not only is cyanide-resistance widespread amongst the plant kingdom but also highly conserved [ I ] . The cDNA encoding the precursor of the alternative oxidase protein has been isolated and characterised from a number of sources including Sauromatum , Arabidopsis , soybean, tobacco, mango and the yeast Hansenula anomala [see 21. Expression of the Arabidopsis alternative oxidase cDNA in E. coli (haem A deficient) is sufficient to support growth in the presence of cyanide [3]. Amino-acid sequence comparison reveals a high degree of homology amongst all species particularly within the two putative membranespanning helical regions and the C-terminal region. Among the plant sequences, only two of the three Cys residues are conserved and comparison with the yeast sequence reveals that only one of these residues is likely to be important for the functioning of the oxidase [4]. Umbach and Siedow found that the alternative oxidase protein exists as a dimer in the mitochondrial membrane which call occur in a disulphide-linked, less active state or in a more active state when the disulphide bond has been reduced. It has been suggested that the reversible nature of the intermolecular disulphide linkage may provide another mechanism, in addition to that observed with organic acids, for regulating enzyme activity in vivo [5]. The ability of the alternative oxidase to reduce oxygen to water suggests that the active site of the oxidase should contain a coupled transition metal center [I]. Previous metal analyses of partially purified alternative oxidase preparations have been inconclusive; Fe, Cu and Mn have each been reported in varying amounts. Attempts to characterise the alternative oxidase using spectroscopic features have also similarly met with little success [I]. Extensive analyses of isolated plant mitochondria poised in a variety of redox states using electron paramagnetic resonance (EPR) spectroscopy displayed neither any resonances unique to plant mitochondria nor ones whose behaviour were indicative of a specific association with the alternative pathway. A partially purified alternative oxidase preparation from Symplocarpus foeridus spadices also showed no indication of EPR resonances in either the resting (oxidised) or the dithionite-reduced states . Interestingly, the Symplocarpus preparation also had no optical absorbance above 350 nm (Umbach, A.L. and Siedow, J.N., unpublished results), which is unusual for a protein that presumably contains a metal centre. The lack of any optical absorption above 350 nm in alternative oxidase preparations plus the capability to reduce 0 2 to H 2 0 is reminiscent of the properties of soluble methane monooxygcnase [6]. Soluble methane mono-oxygenase is a member of the Fe-0-Fe class of metalloproteins [6], the active site structure of which contains a single 0x0-bridged binuclear iron centre with structural features comparable to that found in hemerythrin and thc R 2 subunit of ribonucleotide reductase. In all three of these enzymes. the oxidiscd proteins contain antiferromagnetically coupled high-spin Fe(II1) atoms. In the fully reduced state, the two iron atoms are in the Fe(I1) redox state and remain coupled. As a result, these binuclear iron proteins show no standard EPR signal in either the oxidised or fully reduced states. The three-dimensional structures of three 0x0-bridged binuclear iron proteins (hemerythrin, the R2 subunit of ribonucleotide reductase and the soluble subunit of methane mono-oxygenase) are currently known [see 61, and they show a common set of structural features. While the exact liganding side chains vary among the different proteins, in every case the iron centre is buried within a scaffold that consists of four long (30-35 residues) a-helices organised as a four-helix bundle. With methane mono-oxygenase and ribonucleotide reductase, two of the four helices in the bundle contain the sequence Glu-X-X-His, which is a characteristic of 0x0-bridged diiron proteins [6]. Each of the histidines and one of the two carboxylates serve as monodentate ligands to the iron atoms in the cluster, while the second carboxylate acts as a hidentate ligand, bridging the two iron atoms. The two remaining protein ligands to the iron atoms are provided by carboxylate residues, one in each of the additional two helices of the four-helical bundle. The liganding histidines are further stabilized within the overall structure by hydrogen bonding to the side chain of the residue immediately Nterminal to the glutamate residue within the opposing Glu-X-XHis sequence. An additional feature of the methane monooxygenase and ribonucleotide reductase active sites is the presence of a hydrophobic cavity formed adjacent to the iron center by a series of conserved residues within the four-helix bundle Analysis of the known alternative oxidase proteins indicated that the published higher plant sequences contain two conserved sequences, Glu-GluA-I-His and Asp-Glu-A-H-His, in the carboxy-terminal hydrophilic domain of the protein, beginning just beyond the second membrane-spanning helical region. Both sets of sequences appear in regions showing a high probability for formation of a helices. These two motifs correspond to those found in the 0x0-bridged binuclear iron cluster of methane mono-oxygenase, with the single conservative substitution of a glutamate for an aspartate residue ncxt to the liganding glutamate in the more N-terminal of the two sequences, Using the Sauromutum amino acid sequence, and starting at Glu268, it i s possible to generate a model containing four successive helices of 10 to 11 residues each. In this scheme, the two Glu-XX-His iron binding motifs are located on helices 1 and 4, which are oriented in anti-paralleled fashion, analogous to helices C and F in methane mono-oxygenase. The second helix in this series contains a conserved carboxylate residue (Asp-28 1 in Sauromatum but Glu in most other plants), oriented toward the amino terminus of the helix which would position it to act as a bidentate ligand to iron atom Fel (analogous to Glu-I14 in helix B of methane mono-oxygenase). Missing from this model is a conserved aspartate or glutamate residue in helix 3 that could serve as the analogue to Glu-209 in helix E of methane monooxygenaw and, hence, the final carboxylate ligand to iron atom Fe2. There no obvious alternative conserved residue in helix 3 that might serve in this capacity. However, in ribonucleotide reductase, Asp-84 in helix B, which is the analogue to Glu-I 14 in methane monooxygenase, acts as a bidentate ligand to Fel. If Glu-319 in helix 4 were to doubly co-ordinate to iron atom Fe2 in the alternative oxidase active site in a fashion analogous to that of Asp-84 in ribonucleotide reductase, that would eliminate the need for the additional carboxylate ligand. In conclusion, by identifying conserved residues in the plant alternative oxidase amino acid sequence, it is possible to construct a structural model incorporating an 0x0-bridged dinuclear iron centre held within a four helix bundle, analogous to the iron centre seen within methane mono-oxygenase and ribonucleotide reductase 1. Moore, A.L. & Siedow, J. N. (1991) Biochim. Biophys. Acta