Reaction of E. coli catalase HPII with cyanide as ligand and as inhibitor

• Published in: Biochimica et biophysica acta 1996-12, Vol.1298 (2), p.241-249
• Main Authors: Maj, Mary, Nicholls, Peter, Obinger, Christian, Hillar, Alex, Loewen, Peter C.
• Format: Article
• Language: English
• Subjects: Animals; Bovine liver; Catalase; Catalase - antagonists & inhibitors; Catalase - genetics; Catalase - metabolism; Cattle; Cyanide; Cyanides - metabolism; E. coli; Escherichia coli - enzymology; Heme d; Hydroperoxidase II; Kinetics; Ligand binding kinetics; Ligands; Liver - enzymology; Mutagenesis, Site-Directed; Spectrum Analysis
• ISSN: 0167-4838; 0006-3002; 1879-2588
• DOI: 10.1016/S0167-4838(96)00134-3

Cyanide forms an inhibitory complex with the haem d-containing E. coli catalase HPII, spectrally similar to the cyanide complex of beef liver enzyme but with absorption bands shifted 90 nm towards the red end of the spectrum. Both the K d and K i values are ≈ 7 μM in the wild-type enzyme. The cyanide reaction is slow, with a bimolecular ‘on’ constant approx. 2000 X smaller than that of eukaryotic enzyme, and an ‘off’ constant diminished by a similar amount. Catalases with a mutated distal histidine (H128) fail to bind cyanide at cyanide concentrations below 50 mM. Catalases with a mutated distal asparagine (N201) show only small changes in cyanide affinity from the wild type. The major fraction of HPII N201A has a K d = 40 μM, and a minor fraction has a lower cyanide affinity; the major fraction of HPII N201Q has a K d = 15 μM. The K d and K i for HPII N201D is ≈ 8 μM, essentially identical with that of the wild type but N201D appears to bind cyanide somewhat more rapidly than does wild-type enzyme. The HPII mutant N201H can be obtained in both haem d and protohaem forms; it exhibits two types of cyanide binding behaviour. In its protohaem form it binds cyanide poorly ( K d ≥ 0.25 mM). After peroxide treatment converts it into haem d or a closely related species it binds cyanide with a much higher affinity ( K d = 15 μM). Cyanide binding to HPII requires a distal histidine to provide hydrogen-bonding stability, but not a distal asparagine. Rates of cyanide binding and release are controlled by haem group accessibility through the channel leading to the outside. In HPII N201H channel opening may depend upon oxidation of the haem from the starting protohaem to the final haem d form.