Observation of fast release of NO from ferrous d 1 haem allows formulation of a unified reaction mechanism for cytochrome cd 1 nitrite reductases

Abstract

Cytochrome cd 1 nitrite reductase is a haem-containing enzyme responsible for the reduction of nitrite into NO, a key step in the anaerobic respiratory process of denitrification. The active site of cytochrome cd 1 contains the unique d 1 haem cofactor, from which NO must be released. In general, reduced haems bind NO tightly relative to oxidized haems. In the present paper, we present experimental evidence that the reduced d 1 haem of cytochrome cd 1 from Paracoccus pantotrophus releases NO rapidly (k = 65-200 s -1); this result suggests that NO release is the rate-limiting step of the catalytic cycle (turnover number = 72 s -1). We also demonstrate, using a complex of the d 1 haem and apomyoglobin, that the rapid dissociation of NO is largely controlled by the d 1 haem cofactor itself. We present a reaction mechanism proposed to be applicable to all cytochromes cd 1 and conclude that the d 1 haem has evolved to have low affinity for NO, as compared with other ferrous haems. © The Authors Journal compilation © 2011 Biochemical Society.