The Production of Ammonia by Multiheme Cytochromes c - The Metal-Driven Biogeochemistry of Gaseous Compounds in the Environment - page 223

In a consecutive study, DFT theory was employed to investigate the recharging of
the active Fe heme center 1 (Figure 6 ) with protons and electrons via a series of
reaction intermediates, Fe(II)-NO + ,Fe(II)-NO ￿ , Fe(II)-NO , and Fe(II)-HNO
[ 68 ]. The activation barriers for the various proton and electron transfer steps were
estimated in the framework of Marcus theory. A radical transfer role for the active-
site Tyr218 could not be found in the calculations, whereas the important role of the
highly conserved Ca 2+ ion located in the direct proximity of the active site in proton
delivery was confirmed, in agreement with earlier experimental findings [ 70 ]. Most
recently, the second half-cycle of the six-electron NO 2 ! NH 3 reduction mechanism
was analyzed by Neese and coworkers [ 69 ]. In total, three electrons and four protons
have to be delivered to obtain the final product, NH 3 , starting from the HNO
intermediate. Two isomeric radical intermediates, HNOH ￿ and H 2 NO ￿ , are postulated
which are readily converted to H 2 NOH, most likely through intramolecular proton
transfer from residues Arg114 or His277. After N-O bond cleavage a radical
intermediate H 2 N ￿ is formed which finally reacts with Tyr218, assigning for the
first time a specific role of this amino acid residue in the final step of the nitrite
reduction process, as expected from the earlier mutational investigations [ 72 ].
Obviously, the active site heme 1 accommodates anions and uncharged
molecules, such as NO or hydroxylamine, and releases the NH 4 cation only after
the full six-electron reduction of NO 2 . The preference for anions is reflected by a
positive electrostatic potential around and inside of the active site cavity (Figure 8 ).
Considering the good accessibility of the active site for water molecules and the
presumably lowered pH on the periplasmic side of the cytoplasmic membrane
where nitrite reductase is located (Section 4.3 .), the product of nitrite reduction
will be the positively charged NH 4 ion rather than uncharged NH 3 . The cationic
product can use a second exit channel leading to the protein surface opposite to the
entry channel. It branches before reaching the protein surface and ends in areas with
a significantly negative electrostatic surface potential. The presence of separate
pathways for substrate and product with matched electrostatic potential will con-
tribute to the high specific activity of nitrite reductase (Figure 8 ).
4.3 Electron Transfer Routes to Cytochrome c
Nitrite Reductase
As described in the previous section, NrfA was found to form a stable complex with
the tetraheme cytochrome c NrfH in species such as W. succinogenes and
Desulfovibrio vulgaris . In this arrangement, NrfH anchors the complex in the
membrane and catalyzes menaquinol oxidation as well as electron transport to
NrfA (Figure 9 , top and Table 1 , enzyme classes 2.1 and 2.2) [ 63 , 73 , 74 ]. NrfH
is a member of the widespread NapC/NrfH family [ 24 , 75 , 76 ]. Such proteins are
membrane-bound tetra- or pentaheme cytochromes c that comprise an N-terminal