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Homeostatic control of nitric oxide ( NO ) at nanomolar concentrations in denitrifying bacteria – modelling and experimental determination of NO reductase kinetics in vivo in P aracoccus denitrificans
Author(s) -
Hassan Junaid,
Bergaust Linda L.,
Molstad Lars,
Vries Simon,
Bakken Lars R.
Publication year - 2016
Publication title -
environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.954
H-Index - 188
eISSN - 1462-2920
pISSN - 1462-2912
DOI - 10.1111/1462-2920.13129
Subject(s) - denitrifying bacteria , nitric oxide , in vivo , nitrite , biology , kinetics , nitrate reductase , nitrite reductase , biophysics , biochemistry , enzyme , homeostasis , enzyme kinetics , reductase , denitrification , chemistry , nitrate , microbiology and biotechnology , ecology , nitrogen , active site , endocrinology , physics , organic chemistry , quantum mechanics
Summary Homeostatic control of nitric oxide ( NO ) at nanomolar concentrations appears common among denitrifying bacteria, often ascribed to synchronized expression of nitrite and nitric oxide reductase ( N ir and N or). We questioned whether this is sufficient: using the reported substrate affinities for cytochrome cd 1 nitrite reductase ( c N or), our model of batch cultures of P aracoccus denitrificans predicted NO concentrations orders of magnitude higher than measured. We rejected a hypothesis that the homeostatic control is due to a negative feedback by NO on the activity of N ir S because the inclusion of such feedback resulted in too slow anaerobic growth and N 2 production. We proceeded by determining the kinetic parameters for c N or in vivo by a carefully designed experiment, allowing the estimation of NO concentration at the cell surface while anoxic cultures depleted low headspace doses of NO . With the new parameters for c N or kinetics in vivo { v = v max /[1 + K 2 /( NO ) + K 1 × K 2 /( NO ) 2 ]; v max = 3.56 fmol NO cell −1 h −1 , K 1 < 1 nM, and K 2 = 34 nM}, the model predicted NO concentrations close to that measured. Thus, enzyme kinetics alone can explain the observed NO homeostasis. Determinations of enzyme kinetic parameters in vivo are not trivial but evidently required to understand and model NO kinetics in denitrifying organisms in soils and aquatic environments.