Similar to the iron oxygenases, also this class of enzymes can be subdivided into heme and non heme iron nitric oxide reductases. All of them contain dinuclear iron centers.[83]Heme diiron nitric oxide reductases, or shortly termed NORs, feature a heme iron center termed heme-b3 located in close proximity to a non heme iron center Feb.[84] This structural motif is largely found in denitrifying bacteria. NorBC, cNOR or heme b3-FeB are among other enzymes associated with heme nitric oxide reductase activity and this recurring structural motif.[83] NORs are capable to perform the reduction of two equivalents of NO with two electrons and two protons to N2O. The mechanism of this process is to date not fully elucidated. Synthetic models have been developed to perform binding studies of NO to the heme iron center and to study electronic effects that might influence NO reduction.[85–90] Apart from these heme/non heme NORs another family of enzymes has gained significant interest in literature. The large family of non heme diiron enzymes has been studied for decades with regard to oxygen activation. Today it is known that certain representatives, the Flavodiiron Proteins (FDPs), are especially suitable to reduce aside
from dioxygen also nitric oxide. The following section will discuss this very intriguing group of enzymes in more detail.
1.7.1 Flavodiiron Proteins (FDPs)
Flavodiiron proteins, expressed by some microorganisms to fight the immune response in mammals, were developed to sense and detoxify nitric oxide.[91] Apart from their func-tion of reducing nitric oxide to the less toxic nitrous oxide they are as well able to ac-tivate dioxygen. Literature therefore attributes this class of enzymes a dual functional-ity.[91,92] Generally though, FDPs are either more selective for nitric oxide (from E. coli andSalmonella enterica) or for dioxygen (e.g., eukaryotic FDPs from anaerobic protozoa or from methanogens).[91] Most FDPs contain a Flavin mononucleotide (FMN) cofactor and a non heme diiron site. The first flavodiiron protein was structurally characterized in 2000 from Desulfovibrio gigas as oxido-reductase.[93] The protein is a homodimer. Each monomer is composed of two domains, a β-lactamase unit with the non heme diiron cen-ter and a flavodoxin domain with the FMN cofactor. Within the non heme diiron site an oxygen molecule was located, which led to the conclusion that the discovered protein belonged to the family of diiron oxygenases. A second crystal structure from Moorella thermoacetica FprA (A-type flavoprotein) was published by Lanzilotta and cowork-ers in 2005.[94] Three forms of the FDPs active site were reported: an oxidized form, a dithionite reduced and a dithionite form that had reacted with NO.
Different from the FDP fromDesulfovibrio gigas, two homodimers were found in the struc-ture. Each homodimer though bears, as decribed above, two domains in close proximity containing the FMN cofactor and the non heme diiron site. The diiron core is coordinated by in total four terminal histidines and three carboxylates from glutamate and aspartate.
One bridging solvent molecule completes the coordination sphere. Scheme 1.13 depicts ligands and coordination modes of the diiron site in FprA. The arrow marks the only difference to the structure of the protein from Desulfovibrio gigas where one histidine is being exchanged for a water molecule.[95]
FeIII FeIII
Scheme 1.13: Schematic representation of the diiron active site inMoorella thermoacetica.
The FMN cofactor is assumed to contribute to the fast electron transfer and thus FNOR reactivity of the enzyme.[96]It is located in a proximity of 4-6 Å to the active site. Its exact function in the enzymes’ catalytic cycle though is not known to date.[97]Not known yet is moreover the binding of nitric oxide to the diiron core. The crystallographic structure of FprAin 2005 was obtained of a diiron site without nitric oxide or dioxygen bound. Nitric oxide was modeled to fit in the diiron pocket via a space filling model.[94] With this key
aspect not elucidated, it is not surprising that the catalytic cycle and the process of nitric oxide reduction are under discussion in literature. Among many proposals basically four mechanisms are considered for the following reaction sequence:
2NO + 2e–+ 2H+−−→N2O + H2O (1.2) An overview of the different mechanisms for the reduction of nitric oxide is given in Scheme 1.14.
Scheme 1.14: Selection of proposed mechanisms for the reduction of NO and N2O generation by flavodiiron proteins.[96,98]
In three of the four pathways a diferrous dinitrosyl intermediate is proposed. This in-termediate can be reduced either directly by the addition of protons via the diferrous dinitrosyl pathway, by the sequential reduction mediated by the flavin mononucleotide cofactor and subsequent protonation via the mixed valence mechanism or by an H-atom transfer through the cofactor via a superreduced mechanism. A fourth possibility lies in the formation of a hyponitrite intermediate followed by protonation and N2O release.
Recent studies reacting the dithionite reduced diiron center of FDPs with sequential equiv-alents of NO support the formation of a diiron dinitrosyl species, which would disregard the hyponitrite mechanism.[99]The formation of the dinitrosyl intermediate in this study seems to proceed via a mononitrosyl species first, which has been trapped by stopped flow UV/Vis measurements and EPR studies. The spin state of each iron center in the diiron dinitrosyl species was determined to 3/2 with characteristic Mössbauer parameters. The diiron dinitrosyl sites were found to be antiferromagnetically coupled resulting in anS= 0 ground state. Aside from the observation of these two species the reaction pathway of NO reduction in FDPs is still unknown. Calculations point toward a superreduced
mech-anism[97] whereas experimentally and from model chemistry no option can be reliably ruled out as even the hyponitrite mechanism has been postulated for recent diruthenium dinitrosyl- and tricopper nitrosyl complexes.[100,101]