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

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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.

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Israel Journal of Chemistry

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