Title

Studies on Pseudomonas aeruginosa cd1 nitrite reductase: The association and dissociation reactions of the d1-heme

Document Type

Article

Publication Date

1-1-2000

Abstract

The dissociation and association reactions of the d1-heme, the prosthetic group characteristic of cd1 nitrite reductases (NiRs), have been investigated to assess the stability of the native enzyme. At pH 5.0 and 37 °C, the rate constant for the dissociation of the ferric d1-heme from native NiR purified from Pseudomonas aeruginosa is 4.7 ± 1.4 × 10-4 s-1. However, when the d1-heme is in the ferrous state no dissociation is observed, consistent with the shortening and strengthening of the proximal bond upon reduction of the iron. Recombinant wild-type protein and two single point mutants (Y10N and Y10F), which are expressed as semi-apo proteins and were reconstituted with synthetic d1-heme, display the same slow dissociation rate as the native enzyme. Therefore the stability of the d1-heme bound in the crevice provided by the eight-bladed β propeller domain is not altered by the act of reconstitution or by these two point mutations. The association reaction between the ferric d1-heme and semi-apo NiR is second-order and governed by an apparent rate constant of 3.3 × 106 M-1 s-1 at neutral pH and 25 °C. Interestingly, the combination rate constant is an order of magnitude slower than that reported for iron protoporphyrin IX and apomyoglobins or apohemoglobins. This difference appears to be a property of the d1-heme and not of the protein since association rate constants of CO-protoheme-Fe(II) and dicyanoprotoheme-Fe(III) with semi-apo NiR are 5 × 107 M-1 s-1 and 6 × 107 M-1 s-1, respectively. These results are discussed with reference to the structure of the d1-heme binding site, as inferred from the known 3D structure of P. aeruginosa NiR.

Publication Source (Journal or Book title)

Israel Journal of Chemistry

First Page

27

Last Page

33

This document is currently not available here.

Share

COinS