Bindings of NO, CO, and O to multifunctional globin type dehaloperoxidase follow the 'sliding scale rule'.

Dehaloperoxidase-hemoglobin (DHP), a multifunctional globin protein, not only functions as an oxygen carrier as typical globins such as myoglobin and hemoglobin, but also as a peroxidase, a mono- and dioxygenase, peroxygenase, and an oxidase. Kinetics of DHP binding to NO, CO, and O were characterized for wild-type DHP A and B and the H55D and H55V DHP A mutants using stopped-flow methods. All three gaseous ligands bind to DHP significantly more weakly than sperm whale myoglobin (SWMb). Both CO and NO bind to DHP in a one-step process to form a stable six-coordinate complex. Multiple-step NO binding is not observed in DHP, which is similar to observations in SWMb, but in contrast with many heme sensor proteins. The weak affinity of DHP for O is mainly due to a fast O dissociation rate, in accordance with a longer N-Fe distance between the heme iron and distal histidine in DHP than that in Mb, and an open-distal pocket that permits ligand escape. Binding affinities in DHP show the same 3-4 orders separation between the pairs NO/CO and CO/O, consistent with the 'sliding scale rule' hypothesis. Strong gaseous ligand discrimination by DHP is very different from that observed in typical peroxidases, which show poor gaseous ligand selectivity, correlating with a neutral proximal imidazole ligand rather than an imidazolate. The present study provides useful insights into the rationale for DHP to function both as mono-oxygenase and oxidase, and is the first example of a globin peroxidase shown to follow the 'sliding scale rule' hypothesis in gaseous ligand discrimination.