Pseudoazurin dramatically enhances the reaction profile of nitrite reduction by Paracoccus pantotrophus cytochrome cd1 and facilitates release of product nitric oxide.
Cytochrome cd(1) is a respiratory nitrite reductase found in the periplasm of denitrifying bacteria. When fully reduced Paracoccus pantotrophus cytochrome cd(1) is mixed with nitrite in a stopped-flow apparatus in the absence of excess reductant, a kinetically stable complex of enzyme and product forms, assigned as a mixture of cFe(II) d(1)Fe(II)-NO(+) and cFe(III) d(1)Fe(II)-NO (cd(1)-X). However, in order for the enzyme to achieve steady-state turnover, product (NO) release must occur. In this work, we have investigated the effect of a physiological electron donor to cytochrome cd(1), the copper protein pseudoazurin, on the mechanism of nitrite reduction by the enzyme. Our data clearly show that initially oxidized pseudoazurin causes rapid further turnover by the enzyme to give a final product that we assign as all-ferric cytochrome cd(1) with nitrite bound to the d(1) heme (i.e. from which NO had dissociated). Pseudoazurin catalyzed this effect even when present at only one-tenth the stoichiometry of cytochrome cd(1). In contrast, redox-inert zinc pseudoazurin did not affect cd(1)-X, indicating a crucial role for electron movement between monomers or individual enzyme dimers rather than simply a protein-protein interaction. Furthermore, formation of cd(1)-X was, remarkably, accelerated by the presence of pseudoazurin, such that it occurred at a rate consistent with cd(1)-X being an intermediate in the catalytic cycle. It is clear that cytochrome cd(1) functions significantly differently in the presence of its two substrates, nitrite and electron donor protein, than in the presence of nitrite alone.
Sam
Katharine A
KA
Department of Biochemistry, University of Oxford South Parks Road, Oxford OX1 3QU, United Kingdom.
Fairhurst
Shirley A
SA
Thorneley
Roger N F
RN
Allen
James W A
JW
Ferguson
Stuart J
SJ
D001400:Azurin / Q000494:pharmacology*; D002384:Catalysis / Q000187:drug effects / Q000528:radiation effects; D003574:Cytochrome c Group; D003580:Cytochromes / Q000378:metabolism*; D008027:Light; D009569:Nitric Oxide / Q000378:metabolism*; D009572:Nitrite Reductases / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism* / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects / Q000528:radiation effects; D042084:Paracoccus pantotrophus / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D019163:Reducing Agents / Q000494:pharmacology; D013057:Spectrum Analysis; D015032:Zinc / Q000378:metabolism
D003574:Cytochrome c Group; D003580:Cytochromes; D009573:Nitrites; D019163:Reducing Agents; C047163:pseudoazurin; D001400:Azurin; D009569:Nitric Oxide; C041349:potassium nitrite; C019315:cytochrome cd1; D009572:Nitrite Reductases; D015032:Zinc