H-NOX from Clostridium botulinum, like H-NOX from Thermoanaerobacter tengcongensis, Binds Oxygen but with a Less Stable Oxyferrous Heme Intermediate.

Abstract

Heme nitric oxide/oxygen binding protein isolated from the obligate anaerobe Clostridium botulinum (Cb H-NOX) was previously reported to bind NO with a femtomolar K(D) (Nioche, P. et al. Science 2004, 306, 1550-1553). On the other hand, no oxyferrous Cb H-NOX was observed despite full conservation of the key residues that stabilize the oxyferrous complex in the H-NOX from Thermoanaerobacter tengcongensis (Tt H-NOX) (the same study). In this study, we re-measured the kinetics/affinities of Cb H-NOX for CO, NO, and O2. K(D)(CO) for the simple one-step equilibrium binding was 1.6 × 10(-7) M. The K(D)(NO) of Cb H-NOX was 8.0 × 10(-11) M for the first six-coordinate NO complex, and the previous femtomolar K(D)(NO) was actually an apparent K(D) for its multiple-step NO binding. An oxyferrous Cb H-NOX was clearly observed with a K(D)(O2) of 5.3 × 10(-5) M, which is significantly higher than Tt H-NOX's K(D)(O2) = 4.4 × 10(-8) M. The gaseous ligand binding of Cb H-NOX provides another supportive example for the "sliding scale rule" hypothesis (Tsai, A.-L. et al. Antioxid. Redox Signal. 2012, 17, 1246-1263), and the presence of hydrogen bond donor Tyr139 in Cb H-NOX selectively enhanced its affinity for oxygen.