Many strains of fungi have been shown to denitrify, extending this ability, long believed to be restricted to bacteria, to eukaryotes. In the best-characterized organisms, Fusarium oxysporum
His 2+
His His His Fe2+ Fe
Fe2+
His
His His His Fe2+
N O
N
O Fe3+
His
His His His O
Fe3+
Fully-oxidized Fully-reduced
Intermediate N2O 2 NO
2e + 2H+
Figure 10 Proposed structures of the catalytic site of NOR during turnover.
andClindrocarpon tonkinense, the enzymes are mitochondrial and denitrification is coupled to the formation of ATP.107
The enzymology of NO reduction by fungi is unrelated to the bacterial enzymes, but with up to 40% homology is a member of the cytochrome P450 monooxygenase superfamily, and is specif- ically designated as P450nor.108
The NO reductase of F. oxysporum (FoP450nor) is soluble, monomeric (Mrẳ46,000) and contains one protoheme as prosthetic group. Activity is specific for NADH which functions as a direct electron donor to the enzyme which catalyses the reaction ofEquation (6):
2NO + NADH + H+ N O + NAD + H O2 2
+ ð6ị
F Cl
O
F N
N N N
N N N
N Fe
Fe II
II
ArF ArF
1+
F Cl
O O
F N
N N N
N N N
N Fe
Fe III
III
ArF ArF
1+
2e
Low [NO]
N2O [(5L)FeIII–O–FeIII–Cl] +
Structural model for oxidized NOR catalytic site
[(5L)FeIII... FeIII–Cl]+
Structural model for reduced NOR catalytic site, loss of the Fe–Fe oxo bridge
high [NO]
N2O plus NO2 Stable ferrous nitrosyl species
Figure 11 Functional heme/nonheme diiron complex models for NOR activity.
The EPR spectrum of the enzyme at 4 K exhibits both high-spin (gẳ8) and low-spin (gẳ2.3, 2.25, and 1.91) features, consistent with Cys as the fifth axial ligand of the heme iron. The midpoint potential of 350 mV is significantly lower than other P450 cytochromes. The enzyme has been the subject of extensive spectroscopic studies and crystal structures of the enzyme with NO, CO, and native enzyme have been determined.
8.28.5.1 Structure
The structure the ferric resting state of FoP450nor109shown inFigure 12is essentially the same as has been found for the monooxygenase P450. The heme is located between proximal and distal helices, with the thiolate of Cys352 coordinated as the proximal ligand to the heme iron with a FeS bond distance of 2.17 A˚. Compared with the P450 structures, the distal heme pocket is more open to the solvent, which may account for faster binding rates for CO and NO to P450nor.
The global structure of the ferrous CO complex of FoP450nor is unchanged compared with the ferric resting state.109However, the distribution of H2O molecules in the distal pocket is altered.
Since the ferric NO complex is isoelectronic and isosteric in Fe coordination to the ferrous CO adduct, this difference in H-bonding has been proposed to account for the difference in reactivity between the ferric resting state and the ferric NO adduct towards NADH, which can only reduce the latter species. The importance of this H-bonding network has been shown by mutagenesis of heme pocket residues forming a H-bonded network connecting the active site with the solvent to play a critical role in catalysis.
The structures of the ferric NO adduct of FoP450nor and mutant enzymes have been deter- mined under cryogenic conditions at 1.7 A˚ resolution, allowing the NO binding mode and H-bond network to be defined.110As with CO, the binding of NO does not perturb the global structure of the enzyme. The bound NO is highly ordered in a slightly bent conformation with an Fe—NO angle of 161 and an Fe—NO bond length of 1.63 A˚, consistent with earlier EXAFS data.111The Fe atom is essentially in plane with the heme. The electron density on the Fe-bound NO was proposed to be relatively high, corresponding to a formal electronic state of ‘‘Fe3þ–NO.’’ This assignment of a neutral NO entity is consistent with IR data indicating an N—O stretching band at 1851 cm1and the slow autoreduction rate of the ferric FoP450nor NO adduct.
The higher-resolution structures of the wild-type ferric FoP450nor NO adduct shows the presence of two H-bond networks from the heme pocket to the solvent, only one of which was apparent in the lower resolution structures of the native and ferrous CO adduct. In this structure
Figure 12 Structure of FoP450nor.
a water molecule at the heme end of the networks is 3.31 A˚ from the bound NO and is essential for proton delivery required for NO reductase activity.
8.28.5.2 Mechanistic Studies
In formulating a mechanism for FoP450nor two observations have to be taken into account.
First, activity is not inhibited by CO, implying that the external ligand-free state of the Fe2þis not an intermediate in the catalytic cycle. Second, NADH does not react with the ligand-free Fe2þ state, but does reduce the Fe3þNO adduct, presumably as a consequence of an NO-mediated conformational change in the enzyme.
The optical absorption spectra of the stable Fe2þ, Fe3þ, and Fe3þNO species were determined,112 and stopped-flow rapid scan, flash photolysis, and low-temperature spectroscopic methods have been applied to study the reactions ofEquations (7)–(9). The species P450nor(Fe2þNO) formed in reaction (7) was characterized by stopped-flow as having a diminished Soret absorbance at 434 nm and a single broad band at 558 nm. Formation of this species was complete within the dead time of the stopped-flow apparatus, even at 10C and [NO] of 5mM, allowing a limit fork, the rate constant for formation of this species, to be estimated as larger than 107M1s1.
The rate constant for NO binding to P450nor(Fe3þ) to form P450nor(Fe3þNO) (reaction (8)) was determined by monitoring the decay in absorbance at 434 nm as rebinding occurred following laser photodissociation of the P450nor(Fe3þNO) adduct:
P450nor(Fe ) + NO2+ P450nor(Fe2+ããNO) ð7ị
P450nor(Fe ) + NO3+ P450nor(Fe3+ãNO) ð8ị
P450nor(Fe3+ãNO) + NADH Intermediate? ð9ị The reduction of P450nor(Fe3þNO) by NADH (reaction (9)) was followed by rapid-scan spectroscopy. The reaction resulted in the shift of the Soret from 431 nm to form a transient species with Soret at 444 nm and a broad visible absorption at 544 nm, which then decayed to form a species with a peak at 413 nm. These spectral changes are consistent with the intermediate giving rise to the 444 nm band decaying to form P450norFe3þ. The spectral characteristics of the intermediate formed by reduction of P450nor(Fe3þNO) with NADH (peaks at 445 nm and 544 nm) are different from those of P450nor(Fe2þNO) formed by the reduction of P450nor (Fe3þNO) by dithionite (peaks at 434 nm and 558 nm).
The formation of the intermediate species was [NADH] dependent and a second-order rate constant of 0.9106M1s1 was determined. Under limiting [NO] (equivalent to the enzyme concentration) the decay of the intermediate was independent of [NADH] and a single-order rate constant of 0.027 s1was obtained. This rate is too slow to account for the high turnover rate of the enzyme, and it is apparent that excess NO accelerates this reaction since at higher [NO]
(mM) the spectrum of the intermediate was not observed. Under these conditions the absorb- ance changes corresponded to the direct conversion of P450nor(Fe3þNO) to P450nor(Fe3þ).
Since NADH is a two-electron donor, it was suggested that the intermediate is formed by the two-electron reduction of P450nor(Fe3þNO), formally to the (Fe3þNO)2–state, a species without precedent in hemeoprotein chemistry. This is regarded as the key intermediate precursor leading to N—O bond cleavage and N—N bond formation to form N2O. The electronic structure of the intermediate has been probed by time-resolved resonance Raman spectroscopy.113 The low- frequency regions of P450nor(Fe3þNO) and P450nor(Fe2þNO) spectra showed15/14NO-sensitive lines at 530 nm and 544 nm, respectively. These were absent in the spectrum of the intermediate, but were replaced with a new line at 596 nm that was assigned to aFe—NO stretching mode by comparison with model systems. The Fe—NO was assigned a bent conformation since the isotope shift of 10 cm1is larger than that of 5 cm1shown by a linear Fe—NO unit. From these studies the structure Fe2þ(NO)Hþwas proposed, in which a negative charge delocalized over the bound
NO moiety is stabilized by H-bonding to the ordered water network discussed above. This novel structure enables a reaction mechanism in which a second NO molecule attacks the N atom of the intermediate to form a transient hyponitrite (HONNO) in which N—N bond formation occurs.
This species rapidly decomposes to yield N2O and H2O, the N—O bond cleavage step. Based on these data the authors proposed the scheme shown inFigure 13for the catalytic cycle of P450nor.
The more usual reactivity of NADH in functioning as a hydride donor has led to the modified proposal81 that hydride transfer to a FeII–NOþ complex would form HNO coordinated to the heme. The reaction of NO with this species would form N2O and regenerate the ferric heme site for further catalysis.
8.28.5.3 Model Studies
The formation of N2O at a heme center as in P450nor requires the orientation of two NO entities bound in such a way as to facilitate N—N coupling. Bent nitrosyl ligands (as seen in the struc- ture of the ferric P450nor–NO adduct) are subject to electrophilic attack at the nitrogen lone pair by free NO to form a hyponitrite species. On reduction and protonation this species decom- poses to give (N2O2)2which rapidly decomposes to give N2O and H2O to regenerate PtII. This mechanism, shown in Figure 14, was proposed from earlier kinetic studies of the palladium- catalyzed formation of N2O from NO in aqueous solution with CuCl2as reductant.14,114
Fe3+
NADH H+ +
H2O + N2O
NO
Fe3+
N O+
Fe2+
N O– 2e
H+A
NO
Fe2+
N O-
H+ A
NO Fe2+
HO
H+ A
H+ N NO
A
Figure 13 Proposed reaction mechanism for P450nor.
Pd Cl
N O
CuCl2–
2– –
Cl Cl II
N N
2– –
+ NO Pd
Cl N
O Cl
Cl Pd
Cl N
O O O
Cl
Cl II + CuCl2
+ 2 H + Cl –2
N2O + H2O Pd
Cl
Cl Cl II
2 2 3
+ CuCl Cl CuCl2
+ Cl
III
– –
–
Figure 14 Reaction scheme for the formation of N2O by the heterophilic attack of a bound nitrosyl species.