An EPR investigation of the pH-induced transitions in the nitric oxide complex of Arenicola marina deoxyerythrocruorin

Abstract

The EPR spectra of the nitrosyl derivative of Arenicola marina erythrocruorin are reported. A “nine-line” hyperfine spectrum, characteristic of NO trans to imidazole/nitrogen, is observed at pH 7 and this changes to a “three-line” spectrum both at acid and alkaline pH values. These transitions have pK a values of 4.2 and 10.2, respectively, the latter being identical to that observed by absorbance spectroscopy. The acid titration is cooperative (n∼2) while that centered at pH = 10.2 conforms to a simple, single proton transition. The acid transition is discussed in terms of protonation of the proximal histidine residue while that at alkaline values is thought to reflect the decoupling of the NO and the trans-imidazole by rupture of the Fe 2+-N-imidazole bond brought about by a concerted, pH dependent, conformational change also linked to the aggregation state of the subunits comprising the complex. The NO derivative of erythrocruorin, poised at the alkaline pK a value, was unaffected by addition of effectors such as ATP, DPG, IHP, or calcium.