Steady-state kinetics with nitric oxide reductase (NOR): New considerations on substrate inhibition profile and catalytic mechanism

Abstract

Nitric oxide reductase (NOR) from denitrifying bacteria is an integral membrane protein that catalyses the two electron reduction of NO to N2O, as part of the denitrification process, being responsible for an exclusive reaction, the NN bond formation, the key step of this metabolic pathway. Additionally, this class of enzymes also presents residual oxidoreductase activity, reducing O2 to H2O in a four electron/proton reaction. In this work we report, for the first time, steady-state kinetics with the Pseudomonas nautica NOR, either in the presence of its physiological electron donor (cyt. c552) or immobilised on a graphite electrode surface, in the presence of its known substrates, namely NO or O2. The obtained results show that the enzyme has high affinity for its natural substrate, NO, and different kinetic profiles according to the electron donor used. The kinetic data, as shown by the pH dependence, is modelled by ionisable amino acid residues nearby the di-nuclear catalytic site. The catalytic mechanism is revised and a mononitrosyl-non-heme Fe complex (FeB(II)-NO) species is favoured as the first catalytic intermediate involved on the NO reduction.

EPR spectroscopy of putative enzyme intermediates in the NO reductase and the auto‐nitrosylation reaction of Desulfovibrio vulgaris hybrid cluster protein

“…Furthermore: what is the – initial – ES complex? In analysis of steady‐state kinetics of HCuO‐type NO reductases, the reaction is usually written as (Fig ) in which the first two (presumed reversible) reactions represent binding to one and the same or to two different Fe ions, and in which K 1 and K 2 have been reported to be of comparable magnitude . The corresponding rate equation is …”

After all, plasmalemmal expression of type-1 VDAC can be understood. Phosphorylation, nitrosylation, and channel modulators work together in vertebrate cell volume regulation and either apoptotic pathway

“…There are increasing data pointing to the medical relevance of cell membrane-standing VDAC-1 by its involvement in the pathogenesis of several syndromes, e.g., cancer, cystic fibrosis, Alzheimer's Disease, autism, and malaria (Thinnes and Reymann, 1997 ; Best et al, 2007 ; Scharstuhl et al, 2009 ; Bouyer et al, 2011 ; Thinnes, 2013 , 2014 ; Asmarinah et al, 2014 ; Shoshan-Barmatz et al, 2014 ; Tewari et al, 2014 ; Weiser et al, 2014 ; Zhang et al, 2014 ). These data, from my point of view, give reason for the request to keep plasmalemmal VDAC-1 on the schedule in mammalian VDAC studies.…”

Electronic Origin of Catalytic Nitric Oxide Reduction upon Small Rhodium and Copper Clusters

“…) due to the interaction with the environment of enzyme [14][15][16][17][18]. In supported metal catalysts, NO dimer would not have such a multipoint interaction and be weakly adsorbed to the surface as a neutral or monoanion species [9].…”