The Participation of the Hemes of Flavocytochromeb₂₄₅ in the Electron Transfer Process in NADPH Oxidase

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.