27.5: Cytochrome c oxidase (24b.4)

Cytochrome c oxidase (EC 1.9.3.1) is present in most tissues of living organisms and is the terminal enzyme of the electron transport chain. It reacts directly with molecular oxygen in cellular respiration; it catalyzes the electron transfer from cyto­chrome c to O².

Cyto­chrome c oxi­dase activity in erythro­cytes, platelets, or mononuclear leukocytes (lympho­cytes and mon­ocytes) has potential as a sensitive measure of copper status. Animal studies have generally reported reduced tissue cyto­chrome c oxidase activity as an early sign of copper deficiency(Underwood, 1971). in association with a reduction in the respiratory capacity of mito­chondria, especially in the liver, heart, and brain. Coupling of respiration to phos­phoryl­ation is generally not affected (Linder and Hazegh-Azam, 1996). In some of these animal studies, cyto­chrome c oxidase activity in platelets correlated very strongly with liver copper levels (r = 0.99; p < 0.0001) (Johnson et al., 1993).

Genetic defects in the activity of cyto­chrome c oxidase, whereby activity is only about 50% of normal, may lead to neurological, cardiac, and muscle disease in children (DiMauro et al., 1985; Tulinius et al., 1991; Van Coster et al., 1991). Reduced activity of cyto­chrome c oxidase has also been reported in leukocytes in infants and children with Menkes kinky hair syndrome, a genetic defect in copper meta­bolism (Garnica et al., 1977).

More recently, results of copper depletion-repletion studies have reported that platelet cyto­chrome c oxidase activity may be more sensitive than either serum copper or cerulo­plasmin to changes in copper intake. In Figure 24b.2 for example, the activity of cyto­chrome c oxidase in platelets was significantly lower (p < 0.0001) at the end of the copper-depletion period than at baseline Milne and Nielsen, 1996); levels for many women were below the reference range for comparable healthy women (Table 24b.9)

Table 24b.9 Effect of age, gender, and hormone use on mean cytochrome-c oxidase concentrations, measured in both platelets and mononuclear blood cells. FOC, females taking oral contraceptives; FE, females using estrogen therapy. Significance levels from analysis of variance (ANOVA) are also shown (interactions between gender and decade were nonsignificant in both cases). Data from Milne and Johnson, Clinical Chemistry 39: 883–887, 1993

Age (years)	Group	Platelets U/109 cells	Mononuclear blood cells U/106 cells
20–29	M F FOC	2.75 (10) 2.36 (9) 2.87 (9)	0.33 (9) 0.27 (7) 0.32 (7)
30–39	M F FOC	2.94 (9) 2.75 (9 ) 3.87(10)	0.31 (8) 0.36 (10) 0.39 (11)
40–49	M F	3.52 (9) 3.08 (10 )	0.40 (10) 0.35 (9)
50–59	M F FE	3.89 (9) 3.88 (12) 3.54 (3)	0.35 (9) 0.38 (10) 0.42 (3)
60–69	M F	4.92 (7) 4.56 (9)	0.43 (6) 0.37 (9)
70+	M F	4.51 (6) 3.65 (5)	0.45 (4) 0.39 (7)
ANOVA	Gender Decade	NS 0.0001	NS 0.004

(Milne and Johnson, 1993). Moreover, unlike erythro­cyte Zn,Cu-SOD activity or levels of serum copper and cerulo­plasmin, the activity of platelet cyto­chrome c oxidase showed a positive response to copper repletion, with activity levels returning to about 60% of control values (Milne and Nielsen, 1996).

Cyto­chrome c oxidase activity responded similarly in both platelets and mono­nuclear leukocytes to copper repletion in young women fed a copper-depleted diet. However, baseline values were not measured, so that the response of these indices to depletion cannot be evaluated (Milne et al., 1988).

The activity of cyto­chrome c oxidase in both platelets and mono­nucleated leuko­cytes tends to be higher in older adults, and varies markedly between subjects (Table 24b.9, Milne and Johnson, 1993). The activity in mono­nuclear cells, but not platelets, was affected by oral contra­ceptive use, and only in the 30–39y age group. Adolescents with cystic fibrosis also have lower cyto­chrome c oxidase activity in mono­nucleated leuko­cytes, compared with age and sex matched controls (Percival et al., 1995). The postulated mechanism relates to the alterations in chloride transport in cystic fibrosis, which, in turn, impairs copper transport (Alda and Garay, 1990).

The interaction between copper meta­bolism and cyto­chrome c oxidase activity is complex and mediated by transporters, chaperones and affected by their metals and anions. For a recent review see Cobine et al. (2021).

• Interpretive criteria

Table 24b.9 presents mean cyto­chrome c oxidase activity in platelets and mono­nuclear­ blood cells (lymphocytes and mono­cytes) by age and sex for adults 20 to > 70y. Subjects were healthy, non­pregnant, and non­smokers and were not taking any vitamin or mineral sup­plements, or prescribed medications, with the exception of estrogen.

• Measurement of cytochrome-c oxidase

Platelets and mono­nuclear cells have a shorter lifespan (≈10d) than erythrocytes (≈120d) and therefore activities in these shorter-lived cell types better reflect the metabolically active pool of endog­enous copper. However, the isolation of such specific cell types is time consuming and complex, as noted in Section 15.1.3. Moreover, the enzyme cyto­chrome c oxidase is labile, and between subject variation is large. Hence, before the test can be used in community studies, the assay method must be rigorously standardized. An assay developed by Prohaska and Wells (1974) is often used, as are kits that measure ferro­cyto­chrome to ferri­cyto­chrome colour­imetrically.