Studies on Pseudomonas aeruginosa cd(1) nitrite reductase: The associationand dissociation reactions of the d(1)-heme

The dissociation and association reactions of the d(1)-heme, the prosthetic group characteristic of ed, nitrite reductases (NiRs), have been investiga ted to assess the stability of the native enzyme. At pH 5.0 and 37 degrees C, the rate constant for the dissociation of the ferric d(1)-heme from nati ve NiR purified from Pseudomonas aeruginosa is 4.7 +/- 1.4 x 10(-4) s(-1). However, when the d(1)-heme is in the ferrous state no dissociation is obse rved, consistent with the shortening and strengthening of the proximal bond upon reduction of the iron. Recombinant wild-type protein and two single p oint mutants (Y10N and Y10F), which are expressed as semi-ape proteins and were reconstituted with synthetic d(1)-heme, display the same slow dissocia tion rate as the native enzyme. Therefore the stability of the d(1)-heme bo und in the crevice provided by the eight-bladed beta propeller domain is no t altered by the act of reconstitution or by these two point mutations. The association reaction between the ferric d(1)-heme and semi-ape NiR is seco nd-order and governed by an apparent rate constant of 3.3 x 10(6) M-1 s(-1) at neutral pH and 25 degrees C. Interestingly, the combination rate consta nt is an order of magnitude slower than that reported for iron protoporphyr in IX and apomyoglobins or apohemoglobins. This difference appears to be a property of the d(1)-heme and nor of the protein since association rate con stants of CO-protohem-Fe(II) and dicyanoprotoheme-Fe(III) with semi-ape NiR are 5 x 10(7) M-1 s(-1) and 6x10(7) M-1 s(-1), respectively. These results are discussed with reference to the structure of the d(1)-heme binding sit e, as inferred from the known 3D structure of P. aeruginosa NiR.