A needle in a haystack: the active site of the membrane-bound complex cytochrome c nitrite reductase.
Cytochrome c nitrite reductase is a multicenter enzyme that uses a five-coordinated heme to perform the six-electron reduction of nitrite to ammonium. In the sulfate reducing bacterium Desulfovibrio desulfuricans ATCC 27774, the enzyme is purified as a NrfA2NrfH complex that houses 14 hemes. The number of closely-spaced hemes in this enzyme and the magnetic interactions between them make it very difficult to study the active site by using traditional spectroscopic approaches such as EPR or UV-Vis. Here, we use both catalytic and non-catalytic protein film voltammetry to simply and unambiguously determine the reduction potential of the catalytic heme over a wide range of pH and we demonstrate that proton transfer is coupled to electron transfer at the active site.
Almeida
M Gabriela
MG
REQUIMTE, CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2829-516, Monte de Caparica, Portugal.
Silveira
Célia M
CM
Guigliarelli
Bruno
B
Bertrand
Patrick
P
Moura
José J G
JJ
Moura
Isabel
I
Léger
Christophe
C
D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D001665:Binding Sites; D002384:Catalysis; D045263:Cytochromes a1 / Q000737:chemistry* / Q000302:isolation & purification; D003575:Cytochromes c1 / Q000737:chemistry* / Q000302:isolation & purification; D045285:Desulfovibrio desulfuricans / Q000201:enzymology*; D006418:Heme / Q000737:chemistry*; D009565:Nitrate Reductases / Q000737:chemistry* / Q000302:isolation & purification; D011199:Potentiometry; D011522:Protons
D001426:Bacterial Proteins; D011522:Protons; D006418:Heme; D045263:Cytochromes a1; D003575:Cytochromes c1; D009565:Nitrate Reductases; C054690:nitrate reductase (cytochrome)