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bioRxiv · 10.1101/2025.07.16.665236

Cytochrome b5 reductase 4 efficiently reduces Neuroglobin and Cytoglobin

DeMartino, A. W.·Cunningham, O.·Mulluri, S.·Cassel, D.·Hutar, M.·Ji, L.·Deligiannidis, E.·Kaliszuk, S. J.·Huang, X. N.·Tong, Q.·Yien, Y. Y.·Dent, M. R.·Rose, J. J.·Tejero, J.
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Abstract

Cytoglobin and Neuroglobin are heme-containing proteins expressed in most vertebrates, including mammals, with still not completely defined physiological roles. Most of the putative functions of cytoglobin/neuroglobin, such as oxygen binding or nitric oxide dioxygenation, rely on the heme iron being in the ferrous (Fe2+) oxidation state. Therefore, it is very possible that reducing systems are active in the cell to maintain both proteins in the ferrous state. We have previously shown that the cytochrome b5 reductase isoform 3/ cytochrome b5 system, the canonical reductase of hemoglobin and myoglobin, can reduce cytoglobin at very fast rates, consistent with a possible physiological role. However this reducing system is unable to reduce neuroglobin, which to date lacks a validated, physiologically feasible reducing system. Here we have studied the interaction of cytochrome b5 reductase isoform 4 with cytoglobin and neuroglobin and found that cytochrome b5 reductase 4 can reduce cytoglobin at rates comparable to those observed with cytochrome b5 reductase 3/ cytochrome b5. Remarkably, it can also reduce neuroglobin efficiently. Studying different surface mutations of cytoglobin and neuroglobin we note that some cytoglobin mutations, in particular R84E and K116E decrease reduction rates by more than 10-fold, whereas surface mutations in neuroglobin that were shown to impair the interaction of neuroglobin with cytochrome c (E60K/D73K/E87K) show little effect on the reduction rates. We conclude that cytochrome b5 reductase 4 can supplement cytochrome b5 reductase 3/ cytochrome b5 roles for cytoglobin reduction in vivo and is a strong candidate for a physiological role as neuroglobin reductase.

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DeMartino, A. W., Cunningham, O., Mulluri, S., Cassel, D., Hutar, M., Ji, L., Deligiannidis, E., Kaliszuk, S. J., Huang, X. N., Tong, Q., Yien, Y. Y., Dent, M. R., Rose, J. J., Tejero, J.. 2025-07-20. Cytochrome b5 reductase 4 efficiently reduces Neuroglobin and Cytoglobin. https://doi.org/10.1101/2025.07.16.665236

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