Homeostatic control of nitric oxide (NO) atnanomolarconcentrations in denitrifying bacteria – modelling and experimental determination of NO reductase kineticsin vivoinParacoccus denitrificans

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Abstract

Homeostatic control of nitric oxide (NO) atnanomolarconcentrations appears common among denitrifying bacteria, often ascribed to synchronized expression of nitrite and nitric oxide reductase (Nir and Nor). We questioned whether this is sufficient: using the reported substrate affinities for cytochromecd1nitrite reductase (cNor), our model of batch cultures ofParacoccus denitrificanspredicted 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 NirS because the inclusion of such feedback resulted in too slow anaerobic growth and N2 production. We proceeded by determining the kinetic parameters forcNorin vivoby 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 forcNor kineticsin vivo{v=vmax/[1 + K2/(NO) + K1 × K2/(NO)2];vmax = 3.56 fmol NO cell−1 h−1, K1 < 1 nM, and K2 = 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 parametersin vivoare not trivial but evidently required to understand and model NO kinetics in denitrifying organisms in soils and aquatic environments.

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