Fast dissociation of nitric oxide from ferrous Pseudomonas aeruginosa cd1 nitrite reductase. A novel outlook on the catalytic mechanism.
The heme-containing periplasmic nitrite reductase (cd(1) NIR) is responsible for the production of nitric oxide (NO) in denitrifying bacterial species, among which are several animal and plant pathogens. Heme NIRs are homodimers, each subunit containing one covalently bound c-heme and one d(1)-heme. The reduction of nitrite to NO involves binding of nitrite to the reduced protein at the level of d(1)-heme, followed by dehydration of nitrite to yield NO and release of the latter. The crucial rate-limiting step in the catalytic mechanism is thought to be the release of NO from the d(1)-heme, which has been proposed, but never demonstrated experimentally, to occur when the iron is in the ferric form, given that the reduced NO-bound derivative was presumed to be very stable, as in other hemeproteins. We have measured for the first time the kinetics of NO binding and release from fully reduced cd(1) NIR, using the enzyme from Pseudomonas aeruginosa and its site-directed mutant H369A. Quite unexpectedly, we found that NO dissociation from the reduced d(1)-heme is very rapid, several orders of magnitude faster than that measured for b-type heme containing reduced hemeproteins. Because the rate of NO dissociation from reduced cd(1) NIR, measured in the present report, is faster than or comparable with the turnover number, contrary to expectations this event may well be on the catalytic cycle and not necessarily rate-limiting. This finding also provides a rationale for the presence in cd(1) NIR of the peculiar d(1)-heme cofactor, which has probably evolved to ensure fast product dissociation.
Rinaldo
Serena
S
Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Università di Roma La Sapienza, 00185 Rome, Italy.
Arcovito
Alessandro
A
Brunori
Maurizio
M
Cutruzzolà
Francesca
F
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D005296:Ferrous Compounds / Q000737:chemistry* / Q000378:metabolism; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009569:Nitric Oxide / Q000737:chemistry* / Q000378:metabolism; D009573:Nitrites / Q000737:chemistry* / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D005296:Ferrous Compounds; D009573:Nitrites; D009569:Nitric Oxide; D006418:Heme; D010088:Oxidoreductases; C048228:nitrite reductase (heme)