Ca2+ and the bacterial peroxidases: the cytochrome c peroxidase from Pseudomonas stutzeri

Abstract

The production of cytochrome c peroxidase (CCP) from Pseudomonas ( Ps.) stutzeri (ATCC 11607) was optimized by adjusting the composition of the growth medium and aeration of the culture. The protein was isolated and characterized biochemically and spectroscopically in the oxidized and mixed valence forms. The activity of Ps. stutzeri CCP was studied using two different ferrocytochromes as electron donors: Ps. stutzeri cytochrome c(551) (the physiological electron donor) and horse heart cytochrome c. These electron donors interact differently with Ps. stutzeri CCP, exhibiting different ionic strength dependence. The CCP from Paracoccus ( Pa.) denitrificans was proposed to have two different Ca(2+) binding sites: one usually occupied (site I) and the other either empty or partially occupied in the oxidized enzyme (site II). The Ps. stutzeri enzyme was purified in a form with tightly bound Ca(2+). The affinity for Ca(2+) in the mixed valence enzyme is so high that Ca(2+) returns to it from the EGTA which was added to empty the site in the oxidized enzyme. Molecular mass determination by ultracentrifugation and behavior on gel filtration chromatography have revealed that this CCP is isolated as an active dimer, in contrast to the Pa. denitrificans CCP which requires added Ca(2+) for formation of the dimer and also for activation of the enzyme. This is consistent with the proposal that Ca(2+) in the bacterial peroxidases influences the monomer/dimer equilibrium and the transition to the active form of the enzyme. Additional Ca(2+)does affect both the kinetics of oxidation of horse heart cytochrome c (but not cytochrome c(551)) and higher aggregation states of the enzyme. This suggests the presence of a superficial Ca(2+)binding site of low affinity.

A Distinctive Electrocatalytic Response from the Cytochrome c Peroxidase of Nitrosomonas europaea

“…Taking i lim as an enzymatic velocity, a Michaelis-Menten treatment results in an Eadie-Hofstee plot (Fig. 2, inset B) that reveals values of K m and k cat , which are in general agreement with other values reported for other diheme peroxidases (12,16); K m is found to be 55 M for H 2 O 2 and k cat is estimated to be over 1000 s Ϫ1 . We note that for our data, determination of a value of k cat is achieved through an estimation of electroactive coverage, which is typically calculated through the peak area of nonturnover signals (1-3).…”

Identification of Two Catalases in Azotobacter vinelandii : a KatG Homologue and a Novel Bacterial Cytochrome c Catalase, CCC Av

“…Calcium has been shown to be critical for the activation and multimerization of several BCCPs (10,36,47). A sequence bearing some resemblance to the consensus Ca 2ϩ binding site of the BCCPs (TXPYXHXG) was observed at the C-terminal end of the protein (423-SPPYLHDG-430) (Fig.…”

Structure and mechanism in the bacterial dihaem cytochrome c peroxidases

“…The enzyme from Psn [26] behaves very similarly to that from Pap, but the enzymes from Rhodobacter capsulatus (Rbc) [27] and Pseudomonas stutzeri (Pss) [15] do not require added Ca 2+ for either dimerisation or formation of the activated state. Fulop et al [25] suggest that this may reflect methods of cell breakage, in particular the use of moderately alkaline EDTA during spheroplast formation.…”

“…resonance with g z = 3.0 is due to the P haem with its bishistidine coordination and this resonance shifts to g z = 2.85 in the mixed valence enzyme [12,13]. Related enzymes give similar EPR signatures [14][15][16]. Prior to the crystallographic work on the resting enzyme from Psa [17], the importance of Ca 2+ in the structure and mechanism of bacterial CCP had not been recognised.…”