Cytochrome oxidase structure and function

COX, the terminal enzyme (complex IV) of the respiratory chain, is a member of a superfamily of haem-containing terminal oxidases (Calhoun et al. 1994). COX is an integral protein of the inner mitochondrial membrane and has surfaces exposed to both ‘inside’ (matrix) and ‘outside’ (intermembrane space) aqueous compartments. COX catalyses the exergonic transfer of electrons from reduced cytochrome c to molecular oxygen (41-1"^ + 4e" -> 2H2O), and couples this reaction to proton pumping

across the inner mitochondrial membrane. Four protons are consumed in the reaction and another four are translocated from the matrix to the intermembrane space, so that the overall reaction can be represented as;

4 ferrocytochrome c + SH^mside + O2 ^ 4 ferricytochrome c + 4H\uiside + 2H2O

(Wikstrom, 1977). The human enzyme has a molecular weight of approximately 200 kDa and is composed of 13 polypeptide subunits. The three major subunits (I, II and III) constitute the catalytic core of the enzyme and are encoded by mtDNA. The remaining subunits of COX (IV, Va, Vb, Via, VIb, Vic, Vila, Vllb, Vllc and VIII - nomenclature of Kadenbach et al. 1983) are nuclear-encoded. Human subunits Via and Vila each exist as two isoforms, encoded by separate genes. The nucleotide sequences of all 15 genes encoding structural subunits of COX are known, and most have been localised to chromosomes (Table 1.2). The enzyme has 6 metal centres - 2 haems, 2 copper (Cu) centres, a magnesium (Mg) centre and a zinc (Zn) centre. The Zn and Mg centres do not act as redox centres and their function is unclear (Capaldi, 1990b).

The crystal structure of bovine heart complex IV was described by Tsukihara et al. in 1996 (Figure 1.4). The availability of three-dimensional structural information at atomic (2.8 Â) resolution from this X-ray crystallographic data has improved our understanding of the functioning of the enzyme. COX I has 12 membrane spanning and no extramembrane domains, whilst COX II is anchored to the membrane with an N-terminal helix hairpin and has a large C-terminal hydrophilic domain which protrudes into the intermembrane space over the surface of COX I. COX III has 7 membrane spanning domains and its N-terminus lies on the matrix side of the membrane. COX II and III both interact with COX I within the membrane, but have no direct contact with each other. The redox centres involved in electron transfer

Human subunit MW kd AA Yeast homologue Human Gene Gene localisation of human subunit 1 57 513 1 MTC01 mtDNA H5904-7444 II 25.6 227 II MTC02 mtDNA H7586-8262

III 30 261 III MTC03 mtDNA H9207-9990

IV 17.2 147 V C0X4 16q24.2

Va 12.5 109 VI C0X5A 15q25

Vb 10.6 98 IV C0X5B 2cen-q13

Vla-L 9.6 85 via C0X6A1 12q24.2 or 6p21

Vla-H 9.5 85 Via COX6A2 16p

VIb 10.1 85 VIb C0X6B 19q13.1

Vic 8.6 73 Vila C0X6C 8q22-q23

Vlla-H 6.7 58 VII C0X7A1 19q13.1

Vlla-L 6.7 60 VII COX7A2 6 or 14

Vllb 6.4 56 none C0X7B Xp21.1-q21.33

Vllc 5.4 47 VIII C0X7C 5q14

VIII 4.9 44 (Vila) C0X8 11q12-q13

H=heart isoform; L=liver Isoform

Table 1.2 Genes encoding COX subunits

This table was compiled using Inform ation available in the SW ISS-PRO T

(http://www.expasy.ch/sprot/), NCBI LocusLInk (http://w ww .ncbl.nlm .nlh.gov/LocusLlnk/), G EN ATLAS (http://www.cltl2.fr/GENATLAS/) and MITOP

Vic

Vllb

VIII

Va

Vb

Structure of monomer of bovine cytochrome c oxidase

The diagram was constructed with use of the data published by Tsukihara et a! 1996.

are the two haem A moieties (a and 63) and the two copper centres (Cua and Cub). The haem a and the haem as-Cus binuclear centre are associated with COX I, while COX II contains the Cua centre and accepts electrons from cytochrome c. Electrons are transferred from Cua via haem a to the haem as-Cus binuclear centre, where oxygen reduction takes place on the matrix side of the inner membrane (Hill, 1993). COX III does not contain any prosthetic groups and its removal in some systems does not appear to affect COX activity (Capaldi, 1990a). The function of COX III therefore remains unclear. The exact mechanism of proton pumping by COX is still debated (Gennis, 1998; Michel, 1998). The role of the nuclear-encoded subunits remains obscure, despite the availability of sequence and structural information (Grossman and Lomax, 1997). None of the nuclear-encoded subunits has a

counterpart in any prokaryotic COX and so it seems unlikely that these subunits play a role in electron transfer or proton pumping (Taanman, 1997). It has been

postulated that these subunits may be involved in assembly or stability of the holoenzyme complex, and/or may have regulatory functions by binding ligands that modulate the catalytic function of the enzyme. The identification of tissue-specific isoforms of some nuclear COX subunits is seen as evidence for such a regulatory function. Human subunits Via and Vila have both heart (H) and liver (L) specific tissue isoforms (Kadenbach et al. 1982; Yanamura et al. 1988). Only the L isoform is present in liver but both L and H isoforms are found in heart and skeletal muscle. It has been hypothesised that these tissue-specific isoforms may regulate COX activity to the particular needs of the tissue. However different mammalian species have isoforms for different subunits (Linder et al. 1995). This causes difficulties in envisaging how the isoforms might regulate COX activity.

Functional roles for the human nuclear-encoded subunits might be inferred from X- ray crystallography and biophysical studies of the mammalian enzymes and site- directed mutagenesis of the yeast enzyme. X-ray crystallography of bovine COX has demonstrated that none of the 10 nuclear-encoded subunits impinges directly on the metal centres (Tsukihara et al. 1996). In the crystal structure COX exists as a dimer, with subunits Via and VIb acting as bridging peptides between the

monomers. The function of either or both of these subunits may therefore be to stabilise the dimer. X-ray crystallographic data has also demonstrated that subunit Vb provides the 4 cysteine residues that coordinate the Zn atom, and this is the first confirmed role for a nuclear-encoded subunit (Grossman and Lomax, 1997).

Yeast COX is similar to the human enzyme (Table 1.2), and mutagenesis studies in yeast have provided information about the function, assembly and stability of the enzyme complex. For example the yeast homologue of subunit IV appears to modulate the electron transfer rate (Allen et al. 1995). Proteolytic studies have indicated that subunit IV may play a role in proton pumping by mediating access of protons into the transmembrane proton channel (Grossman and Lomax, 1997). This is a potential mechanism by which subunit IV may regulate COX activity. Disruption of the yeast homologue of subunit Va leads to complete loss of cellular respiration (Poyton et al. 1988) whilst disruption of the yeast homologue of subunit Vb appears to prevent COX assembly (Dowhan et al. 1985). Studies of a null mutant for the yeast homologue of subunit Via have demonstrated that this subunit is not required for full activity of the enzyme. However subunit Via contains one of two ATP binding sites on COX and may be involved in ATP-mediated modulation of COX activity (Anthony et al. 1993; Taanman and Capaldi, 1993). Yeast VIb is required for assembly of a fully active enzyme (Grossman and Lomax, 1997) and may regulate enzyme activity by acting as an inhibitor of the electron transfer reaction (Taanman, 1997). The yeast equivalent of subunit Vic is also necessary for a fully active enzyme (Taanman, 1997). Subunit Vila appears to be involved in assembly of the enzyme, possibly by facilitating correct folding or haem incorporation of subunit I (Taanman, 1997). The yeast homologue of subunit Vllc is not necessary for assembly but is required for optimal COX function. The roles of subunits Vllb and VIII are not known.