pH effects on the haem iron co-ordination state in the nitric oxide and deoxy derivatives of ferrous horseradish peroxidase and cytochrome c peroxidase

Abstract

The spectral (e.p.r. and absorbance) properties of the NO and deoxy derivatives of ferrous horseradish peroxidase (HRP; EC 1.11.1.7) and baker's-yeast cytochrome c peroxidase (CCP; EC 1.11.1.5) were investigated between pH 7 and pH 2; over the same pH range the kinetics for CO binding were also determined. At neutral pH the e.p.r. and absorption spectra of the NO and deoxy derivatives of HRP and CCP are typical of systems in which the haem iron is in the hexaco-ordinated state and the pentaco-ordinated state respectively. By lowering pH, the e.p.r. and absorption spectra of HRP and CCP undergo reversible transitions, with pKa values of 4.1 for the NO derivatives and less than or equal to 3 for the deoxy derivatives of the ferrous forms. By analogy with O2-carrying proteins and haem model compounds, the pH-dependent spectral changes of HRP and CCP were interpreted as indicative of the protonation of the N(epsilon) atom of the proximal histidine residue and of the cleavage of the Fe-N(epsilon) bond. However, the slow second-order rate constant (0.003 microM-1.s-1) for CO binding to deoxy ferrous HRP and CCP does not increase substantially even at pH 2.6, suggesting that changes in the Fe-haem plane geometry, presumably associated with the cleavage of the Fe-N(epsilon) bond, do not affect appreciably the observed ligand association rate constant.

EPR Characterization of Axial Bond in Metal Center of Native and Cobalt-substituted Guanylate Cyclase

“…1 and 2). There were several factors to facilitate the cleavage of the iron-histidine bond, including a repulsive trans effect of NO (27,61) and the protonation of the proximal histidine residue (62,63). The latter was proposed for the proximal histidine release of myoglobin and peroxidases at acidic pH.…”

Differential effects of nitric oxide on peroxidase and H2O2 production by the xylem of Zinnia elegans

“…Similar results were found when using either SNAP as NO donor or TMB as substrate. These results may easily be explained if we take into consideration that NO binds to the inactive ferrous form of peroxidases (Ascenzi et al 1989), thus withdrawing activated enzyme forms from the catalytic cycle. In addition, we cannot discard a possible direct chemical action of NO in scavenging of the free radicals generated during peroxidase action, which would explain the minor differences found between the two substrates.…”

Metastable intermediates in myoglobin at low pH.

“…In addition, the properties of the iron-histidine bond may be monitored, since the ironhistidine stretching mode is present in five-coordinate ferrous hemes (10,11). The previous optical absorption experiments on heme proteins at low pH were done with stopped-flow techniques because most of the proteins are not stable for a long period of time under these conditions (3)(4)(5)(6). It is difficult to measure the resonance Raman spectrum with a stoppedflow apparatus because irradiation of a fixed sample with a laser can give photochemical effects such as ligand dissociation (11) and photoreduction (12).…”

“…Myoglobin (Mb) is one of the simplest heme proteins and has been extensively studied in order to understand the factors that control the binding and release of oxygen, its physiological ligand, as well as CO, which is a convenient oxygen substitute for laboratory studies (1,2). In several reports, the binding of CO to Mb (3,4) and other heme proteins (5,6) has been monitored after a rapid drop in pH. For most Mbs and monomeric hemoglobins (Hbs) (3,4) and for human tetrameric Hb (5), it was found that at low pH (-2.5) a large increase in the CO-binding rate occurs.…”