The Nitric Oxide Reductase Mechanism of Flavo-diiron Proteins

Flavo-diiron proteins (FDPs) contain both a non-heme diiron site and a flavin-mononucleotide binding domain. FDPs are widespread in O₂-sensitive bacteria, archaea and also a few protozoan parasites. FDPs are implicated as oxidative or nitrosative stress protection enzymes. In some organisms, FDPs provide both protections. Biochemical evidence indicates that FDPs are the terminal components of a reductive dioxygen and/or nitric oxide scavenging pathway:

O₂ + 4 e- + 4 H+ → 2 H₂O

2 NO + 2 e- + 2 H+ → N₂O + H₂O

The following research focused on characterizing the FDP NOR mechanism. Steady-state activity assays provide little information about NOR pathway intermediates and thus, rapid-mixing techniques were used to obtain pre-steady state kinetics and spectroscopic features of intermediates along the NOR pathway. The NOR pathway was determined to proceed via an antiferromagnetically coupled diferrous-dinitrosyl intermediate and does not require FMNH₂ to catalyze N-N bond formation. The implication of this work on diiron site reactivity towards NO reduction is discussed.

In addition to the wild-type enzyme, FDP from Thermotoga maritima was mutated so as to replace an iron-coordinating histidine with either a weaker donor (asparagine) or a non-coordinating residue (alanine). Surprisingly, the steady-state and pre-steady-state O₂R and NOR activities did not exhibit significant changes compared to the wild-type protein.