To the Editor:
I would like to make the following response to Dr Babior’s recent review in BLOOD.1
Of course, Dr Babior is fully entitled to his belief that the hemes of flavocytochrome b245 are not involved in the electron transfer reactions of NADPH oxidase, but I fail to follow either his logic or his mathematics.
Specifically, I am puzzled about his calculation regarding the turnover of the heme being insufficient to account for the observed rate of O2− production by neutrophils. My own calculation is as follows.
If the turnover rate of the heme is assumed to be 165/s2(the number he quotes), the turnover per minute is 165 × 60 ≅ 10,000/min. Therefore, 1 pmol of heme should generate approximately 10,000 pmol O2− (10 nmol) per minute. 107 PMN contain approximately 75 pmol of heme and should be capable of generating 750 nmol, O2−/min. In our hands, the maximum rate of O2− formation by PMA-activated PMN is approximately 150 nmol O2− min/107 cells and thus would require the participation of only 20% of the heme present.
Secondly, he uses the argument that the rate of heme reduction anaerobically is 1,000 times too slow to support the rate of aerobic O2− production. This reasoning also surely applies to the anaerobic rate of flavin reduction, which we showed to be also approximately 1,000 times too slow.3 Dr Babior apparently does not consider that this argument applies to both heme and flavin equally. I, on the other hand, think that these observations simply strengthen our original argument that the presence of O2 is required to observe kinetically competent rates of electron transfer.
We are both in complete agreement that kinetic competence is the gold standard, but an enzyme’s kinetics are usually studied in the presence of its substrates. This is clearly not the case when NADPH oxidase is studied anaerobically.
There are several lines of evidence indicating that the heme of cytochrome b558 does not participate in electron transport by the leukocyte NADPH oxidase. In addition to the kinetic evidence cited in my review, (1) Cross et al1-1 found that tying up the heme irons of intact neutrophils with butyl isonitrile had relatively little effect on the respiratory burst and (2) an extensively purified preparation of active NADPH oxidase contained considerable FAD but negligible quantities of heme. All these findings are difficult to reconcile with the idea that the hemes in cytochromeb558 are involved in electron transport by the leukocyte NADPH oxidase. As to the turnover number of 10,000 per minute, the justification for choosing cytochromeb558 as the denominator was the assumption that, by analogy with other heme proteins, the hemes of cytochromeb558 carried electrons. However, the turnover number depends only on the quantity of cytochromeb558 in the preparation used for the measurement of O2− production, not on its activity. The figure would be the same regardless of whether or not the heme was actually an electron carrier.