C-H Bond Cleavage Is Rate-Limiting for Oxidative C-P Bond Cleavage by the Mixed Valence Diiron-Dependent Oxygenase PhnZ

Article abstract of DOI:10.1021/acs.biochem.9b00145

PhnZ utilizes a mixed valence diiron(II/III) cofactor and O₂ to oxidatively cleave the carbon-phosphorus bond of (R)-2-amino-1-hydroxyethylphosphonic acid to form glycine and orthophosphate. The active site residues Y24 and E27 are proposed to

Full text of DOI:10.1021/acs.biochem.9b00145

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Article
C-H Bond Cleavage is Rate Limiting for Oxidative C-P Bond
Cleavage By the Mixed Valence Diiron Dependent Oxygenase PhnZ
Simanga R Gama, Becky Suet Yan Lo, Jacqueline Séguin,
Katharina Pallitsch, Friedrich Hammerschmidt, and David L Zechel
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.9b00145 • Publication Date (Web): 02 May 2019
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For submission to Biochemistry, special issue on Current Topics in Mechanistic Enzymology
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C-H Bond Cleavage is Rate Limiting for Oxidative C-P Bond Cleavage By the Mixed
Valence Diiron Dependent Oxygenase PhnZ
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Simanga R. Gama1, Becky Suet Yan Lo1, Jacqueline Séguin1, Katharina Pallitsch2,
Friedrich Hammerschmidt 2, and David L. Zechel1*
1. Department of Chemistry, Queen’s University, Kingston, Ontario, Canada
2. Institute of Organic Chemistry, University of Vienna, Vienna, Austria
*Corresponding author: dlzechel@chem.queensu.ca
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ABSTRACT
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PhnZ utilizes a mixed valence diiron(II/III) co-factor and O2 to oxidatively cleave the carbon-
phosphorus bond of (R)-2-amino-1-hydroxyethylphosphonic acid to form glycine and
orthophosphate. The active site residues Y24 and E27 are proposed to mediate induced-fit
recognition of the substrate and access of O2 to one of the active site Fe ions. H62 is proposed to
deprotonate the C1-hydroxyl of the substrate during catalysis. Kinetic isotope effects, pH rate
dependence, and site directed mutagenesis were used to probe the rate determining transition state
and the roles of these three active site residues. Primary deuterium KIE values of D(V) = 5.5 ± 0.3
and D(V/K) = 2.2 ± 0.4 were measured with (R)-2-amino- [1-2H1]-1-hydroxyethylphosphonic acid,
indicating that cleavage of the C1-H bond of the substrate is rate limiting. This step is also rate
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D
limiting for PhnZ Y24F, as shown by a significant deuterium KIE value of (V) = 2.3 ± 0.1. In
contrast, a different reaction step appears to be rate limiting for the PhnZ E27A and H62A variants,
which exhibited D(V) values near unity. A solvent KIE of D2O(V) = 2.2 ± 0.3 is observed for PhnZ.
Significant solvent KIE values are also observed for the PhnZ Y24F and E27A variants. In
contrast, the PhnZ H62A variant does not show a significant solvent KIE, suggesting that H62 is
mediating proton transfer in the transition state. A proton inventory study with PhnZ indicates that
1.5 ± 0.6 protons are in flight in the rate determining step. Overall, the rate determining transition
state for oxidative C-P bond cleavage by PhnZ is defined by C-H bond cleavage that is coupled to
deprotonation of substrate C1-hydroxyl by H62.
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INTRODUCTION
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Inorganic phosphate (Pi) is an essential metabolic nutrient. In marine and aquatic environments Pi
concentrations can fall to low nanomolar, and occasionally picomolar, concentrations 1,2. Such low
Pi levels cannot adequately sustain microbial life, which has led to the evolution of enzyme
pathways that enable the utilization of organophosphonic acids (Pns), phosphite, and
hypophosphite as alternative sources of Pi. A number of simple Pn are actively synthesized by
bacteria for incorporation into lipids, exopolysaccharides, and glycoproteins. Such commonly
occurring Pns include 2-aminoethylphosphonic acid, methylphosphonic acid, 2-
hydroxyethylphosphonic acid, hydroxymethylphosphonate, and phosphonoacetate 3-5. These
compounds are in turn scavenged by other bacteria for catabolism into Pi. In order to liberate Pi
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from Pns microbes have developed three distinct mechanisms for cleaving the C-P bond . The
heterolytic mechanism is represented by phosphonatase, phosphonoacetate hydrolase, and
phosphonopyruvate hydrolase, where water or an enzyme nucleophile performs in-line attack on
the phosphorus centre to displace a carbanion. Such enzymes are specific for substrates that have
a carbonyl group in the beta-position relative to the phosphorus centre in order to allow resonance
stabilization of the carbanion leaving group. A homolytic mechanism is utilized by the carbon-
phosphorus lyase pathway. C-P lyase can process a wide array of Pn structures, including those
where the alkyl group is not ‘activated’ for heterolytic cleavage. Although the name implies one
enzyme, C-P lyase comprises multiple enzymatic steps and a multiprotein complex; in E. coli this
pathway is encoded by the 14 cistron phn operon (phnCDEFGHIJKLMNOP). Radical based
cleavage of the C-P bond is performed by the PhnJ component, an outlying member of the radical
SAM superfamily. The third mechanism of C-P bond cleavage is represented by the iron dependent
oxygenases HF130PhnY* and HF130PhnZ (herein referred to as simply PhnY* and PhnZ). PhnY*
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is an α-ketoglutarate/non-heme Fe(II) dependent dioxygenase that catalyzes the stereospecific -
hydroxylation of 2-aminoethylphosphonic acid to form (R)-2-amino-1-hydroxyethylphosphonic
acid [(R)-1] . The C-P bond of (R)-1 is then cleaved by the diiron dependent oxygenase PhnZ
(UniProtKB D0E8I5.1) to form Pi and glycine 7. Genes encoding homologs of PhnY* and PhnZ
are commonly found in bacterioplankton that inhabit marine environments with low nanomolar Pi
concentrations, often at frequencies that rival genes encoding hydrolytic or carbon-phosphorus
lyase pathways 8-10. Recent studies have shown that these other pathways allow utilization of
different organophosphonates as Pi sources. For example, a PhnY* / PhnZ pathway from Gimesia
maris is specific for oxidative catabolism of methylphosphonic acid 11, while the TmpA / TmpB
pathway from Leisingera caerulea is specific for 2-(trimethylamonio) ethylphosphonic acid 12.
PhnZ is a member of a novel family of mixed valence diiron oxygenases (MVDOs) 13. The
founding member of this family is the mammalian enzyme myo-inositol dioxygenase, which
oxidatively cleaves the C1-C6 bond of myo-inositol to form D-glucuronate 14. MVDOs comprise
a subfamily within the histidine-aspartate (HD) motif superfamily of phosphohydrolases.
However, instead of using metal ions to activate water for hydrolysis, MIOX and PhnZ use a pair
of iron atoms in a mixed valence (FeII/FeIII) state to activate O2 and perform 4-electron oxidation
of C-C and C-P bonds. Unlike other diiron oxygenases, such as soluble methane monooxygenase,
the mixed valence state is favoured by MIOX and PhnZ over the diferrous state 13. Moreover, no
external electrons are required for catalysis, as all necessary electrons for restoring the active
Fe(II)/Fe(III) state are obtained from the substrate. The two active site Fe ions, denoted Fe1 and
Fe2, are bound by a set of four histidines and two aspartates that are conserved within the HD-
superfamily. Both MIOX and PhnZ use Fe1 to activate O2 while Fe2 is reserved for substrate
binding. However, PhnZ deviates from MIOX in the presence of three distinct residues, Y24, E27,
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and H62, that are proposed to have roles in catalysis and substrate induced activation of O2 15. Y24
and E27 reside on a flexible loop which responds to binding of (R)-1 to Fe2 (Scheme 1). In the
absence of substrate, Y24 blocks the binding of O2 to a Fe1. Upon binding (R)-1 to Fe2, E27 flips
into the active site to form an electrostatic interaction with the 2-amino group of the substrate. This
results in Y24 being removed from the active site and creates a vacant ligand site on Fe1 for O2.
(R)-1 binds to Fe2 in a bidentate fashion using the C1-hydroxyl and one of the phosphonyl
oxygens. The C1-hydroxyl makes an additional interaction with H62, which appears as a
distinctive 5th conserved histidine in PhnZ homologs. Mutagenesis of H62 and E27 have a
significant impact on PhnZ catalysis, while Y24 is largely dispensable for activity 15.
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Scheme 1
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E27
E27
E27
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Y24
NH3
P
Y24
O
H
NH3
OH
NH3
OH
O2
O
Y24
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O
O
H
O
H
HO
FeII
O
O
HO
(R)-1
P
P
O
O
O
H+
O
O
O
O
O
H
H62
H62
FeIII
FeIII
FeII
FeIII
FeIII
H62
O
H
O
O
H
(Fe2)
(Fe1)
H
I
II
III
The nature of the diiron oxo species formed by PhnZ and the relative free energy barriers
for the bond breaking steps are of great interest due to the stabilities of the C-H and C-P bonds in
(R)-1. The C-H bond of a secondary alcohol as found in (R)-1 is estimated to have a bond
dissociation energy of 94 kcal mol-1 16, while that of a phosphonate C-P bond involving a sp3
hybridized carbon is approximately 85 kcal mol-1 17. Cleavage of unactivated C-H bonds was once
thought to be the preserve of high valent iron oxygen complexes, such as the Fe(IV)=O species
formed by Fe(II)/2-oxoglutarate dependent oxygenases 18, or the dioxygen-Fe(IV)/Fe(IV) species
(intermediate Q) used by soluble methane monooxygenase to cleave the C-H bond of methane 19.
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However, MVDOs such as MIOX and a number of mononuclear non-heme oxygenases have
now been shown or are proposed to cleave C-H bonds using ferric superoxo species 21. Both MIOX
and PhnZ are proposed to initiate their respective reactions with C-H bond cleavage by a
diiron(III/III) superoxo species 13. DFT/QM calculations predict C1-H bond cleavage to be the rate
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limiting step for PhnZ . This is in contrast to MIOX, where freeze-quench EPR spectroscopic
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studies indicate that turnover of an advanced intermediate ‘H’ is rate limiting . However, pre-
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steady state accumulation and decay of the diiron(III/III) superoxo species could be observed on
MIOX using deuterium labelled myo-inositol, implicating this species in C-H bond abstraction.
In this study we examined the mechanism of PhnZ through a combination of deuterium
kinetic isotope effects, solvent isotope effects, site directed mutagenesis, and pH-rate analysis. We
show that C-H bond cleavage is rate limiting for PhnZ during oxidative C-P bond cleavage of (R)-
1. Moreover, we show that H62 mediates proton transfer in the rate determining transition state.
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EXPERIMENTAL METHODS
General
All reagents were purchased from Sigma-Aldrich or BioShop Canada, Inc., unless otherwise
specified, and used without further purification. The Malachite green reagent was prepared as
described by Lanzetta and co-workers 23. Wild type PhnZ and the Y24F, E27A, and H62A variants
were expressed and purified as described previously 15. The synthesis of (R)-1 has been described
previously 15.
1H, 13C and 31P NMR spectra were recorded in CDCl3 or D2O on a Bruker Avance AV III
400 (1H: 400.27 MHz, 13C: 100.65 MHz, 31P: 162.03 MHz) spectrometer. Proton NMR chemical
shifts were referenced to residual CHCl3 (δH 7.24 ppm) or HOD (δH 4.80 ppm). 13C NMR spectra
were referenced to CDCl3 (δC 77.23 ppm) and 31P NMR spectra to external H3PO4 (85%) (δP 0.0
ppm). All 13C NMR spectra were recorded j-modulated. Chemical shifts (δ) are given in ppm and
coupling constants (J) in Hz. Optical rotations were measured at 20°C on a Perkin-Elmer 341
polarimeter in a 1 dm cell. [α]D values are given in 10-1 deg cm2 g-1. Melting points were
determined on a Leica Galen III Reichert Thermovar instrument and are uncorrected. TLC was
carried out on 0.25 mm thick MilliporeSigma plates coated with silica gel 60 F254. Spots were
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visualized by UV light and/or dipping the plate into a solution of (NH4)6Mo7O24 4 × H2O (25.0 g)
and Ce(SO4)2 4 × H2O (1.0 g) in aqueous 10% H2SO4 (500 mL), followed by heating with a heat
gun, if not stated differently. Flash (column) chromatography was performed with Macherey-
Nagel silica gel 60 (230-400 mesh).
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Synthesis of (R)-[1-2H1]-2-amino-1-hydroxyethylphosphonic acid {(R)-[1-2H1]-1}
An overview of the synthesis of (R)-[1-2H1]-1 is shown in Scheme 2.
Scheme 2
O
(TMSO)P(OiPr)2
1. DMSO/(COCl)2
2. Et3N
OH
D D
P OiPr
OiPr
HO D
D
BnO
BnO
BnO
O
(±)-[1-2H1]-4
[1,1-2H2]-2
[1-2H1]-3
H2/Pd/C
O
O
O
HN3/DIAD/PPh3
Ac2O
P OiPr
OiPr
P OiPr
OiPr
HO D
P OiPr
OiPr
HO D
N3
AcO D
HO
N3
pyridine
(±)-[1-2H1]-5
(±)-[1-2H1]-6
(±)-[1-2H1]-7
lipase
(Rhizomucor miehei)
conv. 52%
O
O
O
O
1. H2/Pd/C/HCl
2. HCl 6 M reflux
P OiPr
OiPr
Et3N/H2O
P OiPr
P OiPr
OiPr
P OH
OH
N3
AcO D
N3
N3
H2N
OiPr
3. Dowex 50 H+
D OH
D
HO
D
HO
(R)-[1-2H1]-1
(R)-[1-2H1]-7
(R)-[1-2H1]-6
(S)-[1-2H1]-6
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(±)-Diisopropyl [1-2H1]-(2-benzyloxy-1-hydroxyethyl)phosphonate {(±)-[1-2H1]-4}:24,25 Dry
DMSO (3.125 g, 40.0 mmol, 2.84 mL), dissolved in dry CH2Cl2 (8 mL), was added dropwise to a
solution of oxalyl chloride (2.539 g, 20.0 mmol, 1.72 mL) in dry CH2Cl2 (90 mL) at –78°C under
argon. The resulting clear solution was stirred for 10 min before the addition of [1,1-2H2]-2-
benzyloxy-1-ethanol {[1,1-2H2]-2} (2.369 g, 15.4 mmol), dissolved in CH2Cl2 (11 mL). Stirring
was continued for 20 min before the addition of Et3N (4.048 g, 40.0 mmol, 5.54 mL). The reaction
mixture was allowed to slowly warm to –35°C in the cooling bath. Then, aqueous citric acid (50
mL, 39% w/w) was added and the cooling bath was removed. The organic phase was separated
and washed with aqueous citric acid (3 × 60 mL), water (2 × 60 mL) and sat. aqueous NaHCO3
solution (2 × 60 mL). The organic phase was dried (Na2SO4) and concentrated in vacuo.
Diisopropyl trimethylsilyl phosphite (3.789 g, 15.9 mmol) was added dropwise to a solution of the
crude aldehyde [1-2H1]-3 (1.842 g, 12.2 mmol) in dry CH2Cl2 (37 mL) under argon at room
temperature. The reaction mixture was stirred for 16 h then concentrated in vacuo without heating.
The crude silylated intermediate was purified by flash chromatography (hexanes/EtOAc = 1:1, Rf
= 0.50) to remove residual diisopropyl phosphite. The purified silylated phosphonate (2.941 g, 7.5
mmol, 62%) was dissolved in iPrOH (10 mL). HCl (12 M, 7 drops) was added and the reaction
mixture was stirred at room temperature for 1.5 h. Then, the solution was concentrated and the
residue was purified by flash chromatography (EtOAc, Rf = 0.39) to yield the desired racemic
alcohol (±)-[1-2H1]-4 (2.057 g, 6.5 mmol, 53%) as a colorless oil. 1H NMR (CDCl3, 400.27 MHz):
δ = 7.35-7.22 (m, CHarom, 5H), 4.72 (m symm., 2 × 1H, 2 × CH of iPr), 4.56 (AB-system: A-part:
d, J 11.9 Hz, 1H; B-part: d, J 11.9 Hz, 1H, CH2 of Bn), 3.73 (ABP-system: A-part: dd, J 10.4 Hz,
10.3 Hz, 1H; B-part: dd, J 10.4 Hz, 5.5 Hz, 1H; CH2-CH-P), 3.24 (broadened d, J 10.1 Hz, 1H,
OH), 1.30 (d, J 6.0 Hz, 3H, CH3), 1.29 (d, J 6.2 Hz, 3H, CH3), 1.28 (d, J 6.0 Hz, 3H, CH3), 1.26
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(d, J 6.2 Hz, 3H, CH3); 31P NMR (CDCl3, 162.03 MHz): δ = 20.04 (s); 13C NMR (CDCl3, 100.65
MHz): δ = 137.9 (s, Carom), 128.6 (s, 2 × CHarom), 128.00 (s, 2 × CHarom), 127.95 (s, CHarom), 73.6
(s, CH2 of Bn), 71.6 (d, J 6.9 Hz, CH of iPr), 71.5 (d, J 7.1 Hz, CH of iPr), 70.3 (d, J 6.4 Hz, CH2-
CH-P), 68.0 (dt, J 162.2 Hz, 21.6 Hz, CD-P), 24.31 (d, J 2.8 Hz, CH3), 24.28 (d, J 3.1 Hz, CH3),
24.11 (d, J 2.4 Hz, CH3), 24.06 (d, J 2.5 Hz, CH3).
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(±)-Diisopropyl [1-2H1]-(1,2-dihydroxyethyl)phosphonate {(±)-[1-2H1]-5}: HCl (12 M, 3 drops)
and Pd/C (10% Pd, 0.242 g) were added to (±)-[1-2H1]-4 (2.057 g, 6.5 mmol), dissolved in dry
ethanol (30 mL). Then the flask was filled with hydrogen gas to a final pressure of 3.4 bar and
shaken (using a Parr apparatus) at room temperature for 8 h. The catalyst was removed by filtration
(Celite®, moistened with ethanol) and the solvent was removed in vacuo. The crude product (±)-
[1-2H1]-5 (1.469 g, 6.5 mmol, quant.) was dissolved in toluene (15 mL) and concentrated again. It
was used for the following step without further purification. An analytical sample was purified by
flash chromatography (EtOAc, Rf = 0.10) for characterisation; 1H NMR (CDCl3, 400.27 MHz): δ
= 4.97 - 4.63 (m, 3H, OH + 2 × CH of iPr), 3.93 - 3.76 (m, 3H, CH2 + OH), 1.30 (d, J 6.2 Hz, 6
H, 2 × CH3), 1.29 (d, J 6.2 Hz, 6H, 2 × CH3); 31P NMR (CDCl3, 162.03 MHz): δ = 21.42 (s); 13C
NMR (CDCl3, 100.65 MHz): δ = 71.9 (d, J 7.0 Hz, CH of iPr), 71.8 (d, J 7.1 Hz, CH of iPr), 69.2
(dt, J 160.5 Hz, 21.8 Hz, CD-P), 62.9 (d, J 4.9 Hz, CH2), 24.3 (d, J 3.5 Hz, 2 × CH3), 24.1 (d, J
4.9 Hz, 2 × CH3).
(±)-Diisopropyl [1-2H1]-(2-azido-1-hydroxyethyl)phosphonate {(±)-[1-2H1]-6}: DIAD (0.748
g, 3.7 mmol, 0.73 mL) was added to a solution of Ph3P (0.970 g, 3.7 mmol) in dry toluene (13 mL)
at 0°C under argon. The diol (±)-[1-2H1]-5 (0.649 g, 2.9 mmol) was dissolved in a solution of
hydrazoic acid (1.03 M in toluene, 3.59 mL) and added to the reaction mixture after 5 minutes of
stirring. The cooling bath was removed and the reaction mixture was stirred at 40°C for 2 h before
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the addition of another portion of HN3 (1.03 M in toluene, 1.13 mL). Stirring at 40°C was
continued for 45 min before the addition of dry methanol (10 drops). The reaction mixture was
concentrated and the residue was purified by flash chromatography (EtOAc, Rf = 0.41) to give
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azide (±)-[1-2H1]-6 (0.499 g, 1.9 mmol, 66%) as an oily liquid of acceptable purity; P NMR
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(CDCl3, 162.03 MHz): δ = 28.97 (s, O=PPh3, 10 mol%), 19.08 (s, desired product, 85 mol%), 4.42
(s, HP(O)(OiPr)2, 5 mol%). The product was directly acetylated without any further purification.
(±)-Diisopropyl [1-2H1]-(2-azido-1-acetoxyethyl)phosphonate {(±)-[1-2H1]-7}: The
azide (±)-[1-2H1]-6 (1.10 g, 4.2 mmol) was dissolved in dry pyridine (2.088 g, 26.4 mmol, 2.13
mL). Acetic anhydride (0.898 g, 8.8 mmol, 0.83 mL) was added and the reaction mixture was
stirred at 50°C for 1.5 h. The solvent was removed and water (2 mL) was added to the residue. The
resulting mixture was stirred for 10 min before the addition of more water (10 mL). The resulting
slurry was extracted with CH2Cl2 (3 × 10 mL). The combined organic fractions were washed with
HCl (2 M, 2 × 10 mL), followed by an aqueous NaHCO3 solution (saturated, 3 × 10 mL), dried
(Na2SO4) and concentrated in vacuo. The residue was purified by flash chromatography
(hexanes/EtOAc = 1:1, Rf = 0.29) to give the desired racemic acetoxyphosphonate (±)-[1-2H1]-7
(1.153 g, 3.9 mmol, 93%) as a colorless oil of sufficient purity for the chiral resolution.
Chiral resolution of (±)-diisopropyl [1-2H1]-(2-azido-1-acetoxyethyl)phosphonate {(±)-[1-
2H1]-7}:26 Phosphate buffer (50 mM, pH 7, 30 mL) and lipase (from Rhizomucor miehei, ≥ 20000
U/g from Novozyme, 1.5 mL) were mixed and the pH of the resulting solution was adjusted to 7.0
with acetic acid (0.1 M). Deuterated acetoxy-azidophosphonate (±)-[1-2H1]-7 (1.094 g, 3.7 mmol),
dissolved in a mixture of hexanes (7.5 mL) and tert-butylmethylether (2.5 mL), was added to the
mixture. Acetic acid that was released during the hydrolysis reaction was neutralized with
equimolar amounts of NaOH (0.5 M) using an autotitration unit. The reaction was stopped by
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addition of HCl (2 M) to a final pH of 3.0 at a conversion of 53% (25.5 h reaction time). The
organic phase was separated and the aqueous phase was extracted EtOAc (3 × 10 mL). The
combined organic phases were washed with brine (3 × 10 mL), dried (Na2SO4) and concentrated
in vacuo. The two products were separated by flash chromatography (EtOAc/hexanes = 2:1 
EtOAc, Rf (acetate, EtOAc/hexanes 2:1) = 0.42, Rf (alcohol, EtOAc/hexanes 2:1) = 0.21) to give
the deuterated alcohol (S)-[1-2H1]-6 (0.447 g, 1.8 mmol, 95%) and the acetate (R)-[1-2H1]-7 (0.478
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g, 1.6 mmol, 88%). Acetate (R)-[1-2H1]-7: H NMR (CDCl3, 400.27 MHz): δ = 4.81 - 4.68 (m,
2H, 2 × CH of iPr), 3.58 (broadened d, J 6.4 Hz, 2H, CH2), 2.15 (s, 3H, CH3 of acetyl), 1.35 - 1.29
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(m, 12H, 4 × CH3 of iPr); P NMR (CDCl3, 162.03 MHz): δ = 14.72 (s, desired product, 94
mol%), 14.08 (s, unknown impurity, 6 mol%); 20 = –19.4 {c 1.165, acetone, lit. value 15 of non-
[α]퐷
deuterated compound:
20 = –14.3 (c 1.000, acetone)}.
[α]D
(R)-Diisopropyl [1-2H1]-(2-azido-1-hydroxyethyl)phosphonate {(R)-[1-2H1]-6}: Water (0.8
mL) and Et3N (2.191 g, 21.6 mmol, 3.0 mL) were added to the acetate (R)-[1-2H1]-7 (0.455 g, 1.5
mmol) in dry MeOH (15 mL). The resulting mixture was stirred at room temperature for 19 h,
concentrated in vacuo and the residue purified by flash chromatography (EtOAc/hexanes 2:1, Rf =
0.24) to give (R)-[1-2H1]-6 (0.382 g, 1.5 mmol, quant.) as a colorless oil. 1H NMR (CDCl3, 400.27
MHz): δ = 4.83 - 4.68 (m, 2H, 2 × CH of iPr), 3.50 (ABP-system: A-part: dd, J 13.1 Hz, 6.1 Hz,
1H; B-part: dd, J 13.1 Hz, 7.7 Hz, 1H; CH2), 3.44 - 3.33 (m, 1H, OH), 1.36 - 1.30 (m, 12 H, 4 ×
CH3); 31P NMR (CDCl3, 162.03 MHz): δ = 19.06 (s, desired product, 94 mol%), 17.93 (s, unknown
impurity, 6 mol%).
(R)-[1-2H1]-2-amino-1-hydroxyethylphosphonic acid {(R)-[1-2H1]-1}: The azide (R)-[1-2H1]-6
(0.361 g, 1.43 mmol) was dissolved in dry ethanol (20 mL), HCl (12 M, 0.25 mL) and Pd/C (10%
Pd, 0.103 g) were added. The reaction mixture was hydrogenated (3.5 bar, Parr apparatus) at room
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temperature for 2 h. The catalyst was filtered (Celite®, moistened with ethanol) and the filtrate was
concentrated in vacuo. The residue was dissolved in HCl (6 M, 20 mL) and refluxed (oil bath
130°C) for 7 h. The resulting solution was concentrated and applied to an ion exchange resin
(Dowex 50W H+-form, 50 mL, water as eluent). Ninhydrin positive fractions were pooled and
concentrated to give a colourless solid (0.168 g, 1.2 mmol, 83%); TLC: iPrOH:H2O:NH3
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(25%w/w)= 6:3:2, Rf = 0.15. The degree of deuteration was determined from the H NMR
spectrum to be ≥ 99%. The product was crystallised from water/ethanol (60°C  4°C) to give
colorless needles (0.153 g, 1.1 mmol, 75%); m.p 262-267°C (decomp.); 20= –33.5 {c 0.77, H2O,
[α]D
lit. value 15 of non-deuterated compound:
20 = –36.8 (c 0.987, H2O)}; 1H NMR (D2O, 400.27
[α]D
31
MHz): δ = 3.29 (ABP-system: dd, J 13.3 Hz, 6.4 Hz, 1H; dd, J 13.3 Hz, 6.3 Hz, 1H; CH2); P
NMR (D2O, 162.03 MHz): δ = 15.07 (s); 13C NMR (D2O, 100.65 MHz): δ = 64.87 (dt, J 155.2 Hz,
21.3 Hz, CD-P), 41.36 (d, J 8.9 Hz, CH2).
Enzyme Assays
The rate of reaction of (R)-1 with PhnZ or its active site variants was measured by monitoring the
production of Pi using the Malachite green assay. A concentrated solution of PhnZ (5-20 M) was
pre-incubated with 20 molar equivalents of Fe(SO4)2(NH4)2, 5 molar equivalents of ascorbate, and
1 mM TCEP in reaction buffer (50 mM MOPS-HCl, pH 7.00, 150 mM NaCl, 10% glycerol) for
20 min on ice. Reaction buffer containing 1 mg/ml bovine serum albumin (BSA) and (R)-1 in a
total volume of 300 L was pre-incubated at 25°C for 2 min. The reaction was initiated by the
addition of freshly prepared Fe(II) solution (3 L of 10 mM Fe(SO4)2(NH4)2 in 1 mM HCl)
followed by the pre-incubated PhnZ solution (2-5 L). The final reaction mixture consisted of 43
nM PhnZ, 109 M Fe(SO4)2(NH4)2, 209 M TCEP, 214 nM ascorbate, 1 mg/ml bovine serum
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albumin (BSA), and (R)-1 in a total volume of 300 μL. When assaying PhnZ variants, the
following final concentrations were used: PhnZ Y24F, 0.057 M; PhnZ E27A, 0.571 M; PhnZ
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H62A, 0.514 M. Aliquots of 50 μL were removed from the reaction every 30 s and mixed with
50 μL of the Malachite green reagent and the absorbance of the resulting mixture measured at 635
nm using a plate reader (SpectraMax M2, Molecular Devices Corporation). The concentration of
(R)-1 was varied, at a minimum, from 1/5 to 5  the final KM value determined for PhnZ or its
variants. The Pi concentration produced per unit time was determined from a Pi standard curve.
The initial rate (V0) at each concentration of (R)-1 was obtained by a linear regression of the
measured [Pi] as a function of time. A plot of V0/[E]0 vs the concentration of (R)-1 was fit to the
standard Michaelis-Menten equation to calculate kinetic parameters.
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pH Rate Studies
The pH dependence of kcat/KM for PhnZ and active site variants (Y24F, E27A, H62A) were
determined in the following buffers (50 mM) and pH ranges: sodium citrate (pH 4.75); MES (pH
5.5 -6.5); MOPS (pH 7.0); BTP (pH 7.5 - 9.5) and CHES (pH 9.5 - 10). The activity assay was
carried out as described above. A plot of V0/[E]0 vs the concentration of (R)-1 was fit to the
standard Michaelis-Menten equation to calculate kinetic parameters at each pH value. kcat/KM
values for (R)-1 were plotted against pH and fit to equation 1 27 to obtain maximal values of kcat/KM
and the ionization constants pKa1 and pKa2.
p퐾a2 ― pH
(
pH ― p퐾a1)(
)
퐶 × 10 10
푌 =
(1)
pH ― p퐾a1
+ 1 (10p퐾a2 ― pH + 1)
(
10
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In equation (1) Y represents kcat/KM and C is the maximum value of Y. pKa1 and pKa2 are the
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acidic and basic limb ionization constants, respectively.
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Deuterium Kinetic Isotope Effects
Kinetic parameters were determined for wild type PhnZ and active site variants H62A, Y24F and
E27A using (R)-1 and (R)-[1-2H1]-1 as per the enzyme assay method described. The concentration
of (R)-1 and (R)-[1-2H1]-1 was varied, at a minimum, from 1/5 to 5  the final KM value determined
for PhnZ or its variants. The initial rates with (R)-1 and (R)-[1-2H1]-1 were measured on the same
day using the same PhnZ preparation to minimize differences arising from changes in the specific
activity over time. A plot of V0/[E]0 vs the concentration of (R)-1 or (R)-[1-2H1]-1 was fit to the
standard Michaelis-Menten equation to calculate kinetic parameters. The kinetic isotope effect on
D
D
kcat [denoted (V)] or kcat/KM [denoted (V/K)] were calculated from the ratio of parameters
obtained with (R)-1 to those obtained with (R)-[1-2H1]-1.
Solvent Kinetic Isotope Effect
Solvent KIEs on kcat and kcat/KM were determined by measuring kinetic parameters of wild type
PhnZ and its variants (H62A, Y24F and E27A) in aqueous or D2O buffers with (R)-1 varied
typically from 1/5 to 5  the final KM value determined. The D2O buffer was made by dissolving
MOPS and NaCl in 100% D2O and the pD was adjusted by adding NaOD to pD 7.0. The pD value
was obtained by adding 0.4 to the pH meter reading 28. All reagents were prepared in the aqueous
buffer. The same preparation of PhnZ (or its variants) was used for rate measurements in H2O and
D2O buffers. Kinetic parameters were determined by fitting the data to the Michaelis-Menten
equation.
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A proton inventory was performed by measuring activity of wild type PhnZ in buffer with
variable mole fraction of D2O [denoted n(D2O)]. nD2O is defined as the volume of D2O-based
buffer per volume of H2O-based buffer. The relative rate vn/v0, where vn = V0/[E]0 at n mole fraction
of D2O and v0 = V0/[E]0 in the 100% H2O-based buffer, was plotted against n(D2O). The data was
fit to equation 2 29 where n = n(D2O) and s is the number of exchangeable protons responsible for
an observed solvent KIE.
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푠
푣푛
푣0
푛
푆퐾퐼퐸
= 1 ― 푛 +
(2)
(
)
Viscosity Effects
The effect of diffusion-controlled substrate binding and/or product release on the rate of catalysis
was examined by measuring rate as function of solvent viscosity at 1 mM (R)-1. Glycerol was used
as the viscogen to a final a final composition of 0%, 1%, 5%, 10% 15%, 20% (v/v) in reaction
buffer. The relative rate was calculated as v0/vn, where v0 = V0/[E]0 measured in buffer with no
glycerol and vn = V0/[E]0 measured at a given concentration of glycerol in the reaction buffer. v0/vn
was plotted against % glycerol.
RESULTS
Kinetic Analysis
The reaction rate of PhnZ with (R)-1 was measured in a stopped-assay format using Malachite-
green to quantify the Pi release. The PhnZ reaction with (R)-1 was also optimized, affording greater
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activity than what was reported in our earlier work . Key changes to the assay include pre-
incubation of PhnZ on ice with freshly prepared iron(II) ammonium sulfate and the addition of
ascorbate to the reaction mixture (Figure S2). Under these optimized assay conditions, the reaction
of PhnZ with (R)-1 afforded typical Michaelis-Menten kinetics (Figure 1A, black curve). New
kinetic parameters for wild type PhnZ and the active site variants Y24F, E27A and H62A are given
in Table 1. The new PhnZ kcat value is 36-fold greater than reported previously 15. Substitution of
the proposed general acid/base residue H62 for alanine has a major impact on PhnZ activity,
resulting in a 20-fold decrease in kcat, a 4.9-fold increase in KM, and a 100-fold decrease in kcat/KM.
An even greater impact is observed upon substituting the substrate recognition residue E27 for
alanine, affording a 27-fold decrease in kcat, a 37-fold increase in KM, and a 1037-fold decrease in
kcat/KM. However, as observed in our earlier studies 15, substitution of the Fe1 binding residue Y24
with phenylalanine has a modest impact on activity, affording a 1.5-fold decrease in kcat, an 8.9-
fold increase in KM, and a 13.9-fold decrease in kcat/KM. Overall these kinetic parameters are
consistent with our previous proposals 15, where E27 and H62 provide substantial contributions to
transition-state stabilization through direct contact with (R)-1, while Y24 is less important for
catalysis due to its proposed role in controlling access of O2 to Fe1.
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Figure 1. Primary deuterium and solvent kinetic isotope effect measurements with PhnZ. (A) A
plot of V0 / [E]0 versus concentration of (R)-1 (black) or (R)-[1-2H1]-1 (red). (B) A plot of V0 / [E]0
versus (R)-1 in H2O buffer (blue) and 97% D2O buffer (maroon).
Deuterium Kinetic Isotope Effects
To probe whether C-H bond cleavage is rate limiting in the PhnZ reaction, a non-competitive
deuterium kinetic isotope effect (D-KIE) was measured by comparing the kinetic parameters for
(R)-1 and (R)-[1-2H1]-1 (Figure 1A). The kinetic parameters of PhnZ using (R)-[1-2H1]-1 as a
substrate are given in Table 1. Large D-KIE values of D(V) = 5.5 ± 0.3 and D(V/K) = 2.2 ± 0.4 are
observed with wild type PhnZ, indicating that C-H bond cleavage is rate-limiting. Kinetic
parameters for (R)-[1-2H1]-1 were also determined for the H62A, Y24F and E27A PhnZ variants
(Table 1, Figure S1). Interestingly, a negligible D(V) value is observed for PhnZ H62A, but a large
D
D(V/K) is still observed [D(V) = 0.9 ± 0.1; (V/K) = 11 ± 2]. In the case of PhnZ E27A, negligible
D
D
values are observed for both (V) and (V/K). Finally, PhnZ Y24F exhibits a reduced but still
significant D(V) value relative to the wild type enzyme [D(V) = 2.3 ± 0.1] but a negligible D(V/K)
value.
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Solvent Kinetic Isotopic Effects and Proton Inventory
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A solvent KIE was measured for wild type PhnZ by comparing the kinetic parameters for (R)-1 in
water and 97% D2O (pH 7.0) (Figure 1B), affording D2O(V) = 2.2 ± 0.3 and D2O(V/K) = 1.3 ± 0.3
(Table 1). The kinetic parameters for (R)-1 in water and 97% D2O were also determined for the
active site variants H62, Y24F and E27A (Figure S1). This afforded D2O(V) = 0.81 ± 0.05 and
D2O(V/K) = 0.98 ± 0.22 for H62A, D2O(V) = 1.9 ± 0.1 and D2O(V/K) = 2.3 ± 0.05 for Y24F, and
D2O(V) = 5.6 ± 1.7 and D2O(V/K) = 1.5 ± 1.1 for E27A (Table 1). The significant D2O(V) values
observed for wild type PhnZ, Y24F, and E27A reflect transfer of one or more protons in the rate
determining step, while the absence of a significant D2O(V) value with PhnZ H62A suggests that
H62 mediates transfer of one or more protons.
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A proton inventory was performed with wild type PhnZ to provide greater insight into the
observed solvent KIE. The proton inventory experiment was conducted on kcat by measuring initial
rates at one saturating concentration of (R)-1 (1 mM) in various mole fractions (n) of D2O, which
was derived by combining appropriate volumes of D2O and H2O based buffers (Figure 2A). The
plot of vn/v0 versus mole fraction of D2O was fitted to equation 2 29 affording a calculated solvent
KIE value of 2.9 + 1.4 and number of protons in flight s = 1.5 ± 0.6 . This calculated solvent KIE
value is in good agreement with the D2O(V) value measured for PhnZ from the Michaelis-Menton
plots in H2O and 97% D2O. The calculated s = 1.5 ± 0.6 implies that one to two exchangeable
protons are involved in the observed solvent KIE.
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Figure 2. Proton inventory and viscosity measurements with wild type PhnZ (1 mM (R)-1, 25 °C,
pL = 7.00). (A) Proton inventory measurements: a plot of Vn/V0 versus mole fraction of D2O where
Vn and V0 represent V0/[E]0 in a buffer with n mole fraction of D2O and in a buffer with no D2O,
respectively. The data was fitted to equation 2 (solid line) affording a calculated solvent KIE of
2.9 ± 1.4, and number of protons in flight s = 1.5 ± 0.6. (B) Solvent viscosity measurements: a plot
of V0/Vn versus glycerol concentration (%, v/v) where V0 and Vn are V0/[E]0 values in a buffer with
no glycerol and in a buffer with n % glycerol, respectively. The dashed lines are for reference,
where a slope of zero represents a reaction that is not diffusion limited, whereas a slope of 1
represents a reaction that is diffusion limited. A linear fit (solid line) produced a slope of 0.027 ±
0.007.
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Dependence of PhnZ Reaction Rate on Viscosity
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The dependence of the PhnZ reaction rate on solvent viscosity was analyzed to determine whether
diffusion-controlled substrate binding and/or product release limit the rate of catalysis. The effect
of solvent viscosity on kcat was tested by measuring initial rates at saturating concentrations of (R)-
1 (1 mM) at different concentrations of glycerol. The ratio v0/vn was plotted against glycerol
concentration where v0 and vn are initial velocities at zero and n% glycerol, respectively (Figure
2B). A slope of zero is expected if catalysis is not limited by diffusion whereas a slope of unity
represents a reaction that is fully diffusion-limited by nonchemical steps 30,31. For PhnZ an
essentially negligible dependence of kcat on viscosity (m = 0.027 ± 0.007) is observed. This
indicates that the PhnZ reaction is not limited by diffusion of substrate or products from the active
site. Additionally, the viscosity of 20% glycerol (1.76 Centipoises at 20 ºC) 32, the maximal value
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tested with PhnZ, exceeds that of 100% D2O (1.25 Centipoises at 20 ºC) , indicating that the
observed D2O(V) value observed with PhnZ is not an artifact of the greater viscosity of D2O relative
to water.
pH rate dependence of PhnZ and active site variants
The pH dependence of kcat, KM, and kcat/KM for PhnZ and the active site variants Y24F and H62A
were determined by Michaelis-Menten analyses at various pH values. The pH rate dependence of
PhnZ E27A could not be measured with confidence due to the low activity and high KM value of
this variant (Table 1). The pH dependence of kcat/KM for wild type PhnZ and PhnZ Y24F exhibited
bell-shaped profiles with optimums spanning pH 7 that could be fit to an equation describing two
ionizations (Figure 3A and C). Essentially identical apparent pKa values for wild type PhnZ (pKa1
= 5.77 ± 0.05 and pKa2 = 8.85 ± 0.04) and PhnZ Y24F (pKa1 = 5.5 ± 0.2 and pKa2 = 8.7 ± 0.3) are
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observed (Table 2). In the case of PhnZ H62A, the pH dependence of the kinetic parameters could
only be fit with confidence to a single ionisation (pKa = 8.8 ± 0.2, Figure 3B) due to the low activity
and instability of this variant at low pH values.
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pH
Figure 3. pH – rate dependence of PhnZ and its variants with (R)-1. Plots of kcat / KM vs. pH for:
(A) wild type PhnZ; (B) H62A variant and (C) Y24F variant. The kinetic parameters for these
experiments are summarized in Table 1.
DISCUSSION
In this study we show that the oxidative C-P bond cleaving reaction catalyzed by PhnZ is rate-
limited by C-H bond cleavage. The large D-KIE values observed for PhnZ [D(V) = 5.5 ± 0.3] using
(R)-1-[2H1]-1 as substrate indicate that cleavage of the C1-H bond of (R)-1 is the rate-limiting step.
Additionally, the significant solvent KIE observed for PhnZ [D2O(V) = 2.2 ± 0.3] and a proton
inventory indicates that one or two protons are in flight during C-H bond cleavage. One of the
proton transfer events was assigned to H62 on the basis that PhnZ H62A does not show a
significant solvent KIE. Interestingly, a negligible D(V) value accompanied by a large D(V/K) value
is observed with the H62A variant. This implies that the rate limiting step for PhnZ H62A is no
longer C1-H bond cleavage, but instead represents the first irreversible chemical step 34.
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H62 is notable as a conserved 5th histidine residue that is unique to the PhnZ sub-clade of
the HD superfamily of phosphohydrolases (the other four conserved histidine residues are
dedicated to Fe ion binding). H62 is observed by X-ray crystallography to hydrogen bond to the
C1-OH of (R)-1 13,15, and deletion of this side chain has a large effect on kcat and kcat/KM (Table 1),
indicating an important role in the rate determining transition state. Overall, these observations
suggest H62 is acting as a general acid or base during C-H bond cleavage of (R)-1, possibly by
mediating proton transfer to or from the C1-OH. Our results are consistent with the DFT/QM
calculations on PhnZ by Zhao and co-workers 22, which predict that abstraction of the C1 hydrogen
by a Fe1 bound superoxo species is rate limiting, and that H62 acts as a general base in this step
by deprotonating the C1-OH. (Scheme 1, III). Bollinger, Jr. and co-workers have proposed that
one advantage conferred by the mixed-valent diiron (II/III) cofactor over mononuclear iron (II) or
diiron (II/II) cofactors is that the ferric ion can be used to promote ionization of the substrate
hydroxyl while the ferrous ion remains available to activate O2 35. The developing oxyanion
character in the transition state can be expected to stabilize development of a radical at C1
analogous to a ketyl resonance. Steigerwald and co-workers have estimated that such oxyanion
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character can reduce neighbouring C-H bond dissociation energy by 10 to 16.5 kcal mol-1 36
.
Similarly, by deprotonating the substrate C1-OH, H62 can assist in weakening the substrate C-H
bond in the transition state.
As founding members of MVDO family, PhnZ and MIOX form an interesting comparison
due to their application of a conserved active site architecture to cleaving C-P and C-C bonds.
There are notable differences in substrate interactions between the two enzymes. Although the
substrates for PhnZ and MIOX both adopt a bidentate mode of binding to Fe2 in their active sites
(Scheme 3), the ionization states of the C1-hydroxyls appear to be different at the onset of catalysis.
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Instead of histidine, MIOX uses a lysine residue (K127) to interact with C1-OH of myo-inositol
37. Additionally, the C1-OH of myo-inositol is observed to bind to the diiron cofactor of MIOX as
an alkoxide 38,39, which is proposed to be stabilized by the Lewis acidity of Fe2 and the positive
charge of K127 22,37. Therefore, while PhnZ is proposed to perform C-H bond cleavage
concurrently with ionization of the C1-hydroxyl in the transition state (Scheme 3A, I), MIOX
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appears to initiate C-H bond cleavage directly from a C1-alkoxide (Scheme 3B, I) 20,22
.
Scheme 3
A
NH3
O
NH3
NH3
P
NH3
O
O
O
O
O
H
O
P
P
P
RDS
O
H
O
O
O
O
O
O
O
O
O
O
O
O
O
O
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O
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H
H
H
O
H
H
H62
H62
FeIII
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FeII
H62
FeIII
FeIII
FeIII
FeIII
H62
FeII
O
H
O
H
I
O
H
II
III
IV
HO
OH
OH
HO
O
OH
OH
HO
O
OH
OH
HO
OH
OH
B
HO
OH
HO
OH
HO
OH
HO
OH
RDS
(calc)
O
O
H
O
H
O
O
O
O
O
O
O
H
O
H
H
FeII
FeII
FeII
FeIV
NH3
FeIII
FeIII
FeIII
NH3
NH3
NH3
FeIII
O
H
O
H
O
H
K127
K127
K127
K127
I
II
III
IV
The rate limiting step for MIOX also differs from PhnZ. Under steady state conditions, the
reaction of mouse MIOX with [2H6]-myo-inositol does not show a significant D-KIE 20. Instead,
stopped-flow absorbance and freeze-quench EPR studies with MIOX have shown that turnover of
an unknown intermediate ‘H’ is rate limiting 20,35. Calculations by Hirao and co-workers 40 predict
that O-O bond cleavage of a Criegee-like intermediate (Scheme 3B, III), possibly intermediate ‘H’
above, is rate-limiting for MIOX, with an activation barrier 10 kcal mol-1 greater than the C-H
bond cleaving step. Using [2H6]-myo-inositol as a substrate, freeze quench EPR studies were
successful in following the pre-steady-state formation and decay of the diiron Fe(III)/Fe(III)-
superoxo complex that performs C1-H abstraction (Scheme 3B, I to II) 20. From this experiment
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a D-KIE value of  5 was estimated for the C-H cleavage step of MIOX, which is comparable to
the value measured for PhnZ. Interestingly, pig MIOX shows deuterium (kH/kD = 2.1) and tritium
(kH/kT = 7.5) KIEs under steady-state conditions when assayed with [1-2H1] and [1-3H1] labelled
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myo-inositol, respectively , suggesting that under some reaction conditions C-H bond cleaving
step can be partly rate limiting for MIOX.
The differing roles of H62 in PhnZ and K127 in MIOX are also predicted to impact the
fragmentation mechanisms of the Criegee-intermediates formed by each enzyme 22. Calculations
predict that K127 maintains a positive charge throughout the reaction cycle of MIOX, whereas
H62 of PhnZ alternately functions as a general base and acid 22,40. In the case of MIOX, the inability
of K127 to transfer a proton to the Criegee alkoxide promotes homolytic O-O cleavage, leading to
a ferryl species on Fe1 and a gem-diolate alkoxyl radical (Scheme 3B, III to IV). With PhnZ,
protonation of the Criegee alkoxide by H62 is predicted to promote an ‘inverse heterolytic’ mode
of O-O cleavage, leading to a diiron bound orthoester intermediate (Scheme 3A, III to IV). The
comparable O-O homolytic pathway in PhnZ is calculated to be significantly higher in energy (8.8
kcal/mol), likely due to the formation of a high energy ferryl species. In this context it is notable
that C-H bond cleavage is not rate-limiting for the PhnZ H62A and E27A variants, as shown by
the negligible D(V) values but remains at least partly rate-limiting for the Y24F variant [D(V) = 2.3
± 0.1] (Table 1). This suggests that H62 and E27 contribute substantially to another step along the
reaction coordinate, which becomes rate-limiting when these residues are absent. Intriguingly, the
E27A variant still shows a substantial solvent KIE, while the H62A variant does not (Table 1).
This indicates that the rate-limiting step for E27A also involves a proton-transfer event. If the same
step is rate-limiting for E27A and H62A, this suggests that H62 is also mediating proton transfer
in this step.
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Considering their respective roles in Fe1 ion binding, substrate binding, and general acid-
base catalysis, the ionization states of Y24, E27, and H62 can be expected to have an impact on
catalysis. PhnZ exhibits a bell-shaped profile for the dependence of kcat/KM with apparent pKa
values of 5.5 and 8.8 (Table 2). The Y24F and H62A also have bell shaped pH profiles that are
defined by essentially identical apparent pKa values. Unfortunately, an acidic pKa value for H62
could not be unambiguously assigned. The H62A variant could only be assigned a single ionization
with pKa = 8.8. However, due to low activity of H62A, and the instability of this variant at low
pH, the absence of an ionization at low pH cannot be excluded. Overall, the pH dependence of the
reaction of PhnZ with (R)-1 appears to be a composite of multiple ionization events. This would
include the ionization state of the 2-amino group and phosphoryl oxygens of (R)-1, which would
affect interactions with E27 and Fe2, as well as the residues that form a polar pocket (K108, S126,
T129, Q133, and R158) that engage the phosphono group of (R)-1 15.
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The D-KIE values suggest that PhnZ has a high forward commitment to catalysis. This is
reflected in the smaller D(V/K) value [D(V/K) = 2.2 ± 0.4] relative to the D(V) value [D(V) = 5.6] 42.
The forward commitment of an enzyme to catalysis is reflected by the relative magnitudes of the
isotopically sensitive rate constant (C-H bond cleavage in the case of PhnZ) relative to the net rate
constant for dissociation of the reactive enzyme substrate (ES) complex into free enzyme and
substrates 42,43. Because V/K reflects all ES binding interactions up to the first irreversible chemical
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D
step , while V reflects the rate limiting step, suppression of the (V/K) value from the intrinsic
value reflects a slow release of substrate(s) from the active site 42. As illustrated in Scheme 1, V/K
for PhnZ would include contributions from the following sequence of steps: (1) binding of (R)-1,
(2) flipping of the Y24/E27 loop, (3) binding and reduction of O2 by Fe1, (4) C-H bond abstraction
by the Fe1 superoxo species. With PhnZ, the steps involving movement of the Y24/E27 loop and
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subsequent reduction of O2 to superoxide could contribute to a high commitment of catalysis by
hindering dissociation of the reactive PhnZ substrate complex (Scheme 1, III) to free enzyme, O2,
and (R)-1. It should be noted that O2 binding to MIOX is observed to occur reversibly 20, although
this enzyme does not use a direct induced-fit mechanism for substrate binding like PhnZ.
Beyond PhnZ and MIOX, there are relatively few characterized examples of C-H bond
cleavage by ferric superoxo species. The mononuclear non-heme dependent enzyme isopenicillin-
N synthase has been shown to use a ferric superoxo species for abstraction of the C3 hydrogen of
the cysteine moiety of -(-aminoadipoyl)cysteinylvaline 44, a step that is associated with a large
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D-KIE value ranging from 6
to 33 . The related mononuclear non-heme iron dependent
dioxygenases HEPD and MpnS performs two hydrogen abstractions during oxidative C-C bond
cleavage of 2-hydroxyethylphosphonate, the first cleavage of a C-H bond by a ferric superoxo
species, the second cleavage of an O-H bond by a ferryl species 46. The C-H bond cleaving step is
not rate limiting for HEPD or MPnS as a negligible D-KIE is observed on V/K for 2-[2H2]-2-
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hydroxyethylphosphonate; however significant D-KIE and O-KIE values observed on V/K for
O2, indicating reversible formation of the ferric superoxo species, and possibly hydrogen
tunnelling during transfer 46,47. The copper dependent enzymes dopamine ß-monoxygenase,
tyramine ß-monoxygenase, and peptidyglycine -amidating enzyme are also proposed to
reversibly form Cu(II)-superoxo species that perform rate limiting C-H bond cleavage 48. In this
context the large D(V) value observed with PhnZ presents an excellent opportunity to accumulate
high levels of the diiron(III/III) superoxo species for spectroscopic characterization 49.
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ACKNOWLEDGEMENTS
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DLZ thanks the Natural Sciences and Engineering Research Council (NSERC) of Canada for
financial support (492945- 2016 and 03695-2016). KP and FH thank the Austrian Science Fund
(FWF) for support (P27987-N28).
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SUPPORTING INFORMATION
Deuterium and solvent kinetic isotope analysis of PhnZ H62A, Y24F, and E27A (Figure S1, Table
S1), PhnZ rate dependence on iron and ascorbic acid concentrations (Figure S2), and 1H, 13C and
31P NMR spectra of all synthesized compounds.
ACCESSION CODES
PhnZ, UniProtKB: D0E8I5.1
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REFERENCES
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(1) Hudson, J. J., Taylor, W. D., and Schindler, D. W. (2000) Phosphate concentrations in lakes.
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ocean. Science 337, 1104–1107.
(4) Repeta, D. J., Ferrón, S., Sosa, O. A., Johnson, C. G., Repeta, L. D., Acker, M., DeLong, E. F.,
and Karl, D. M. (2016) Marine methane paradox explained by bacterial degradation of
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(5) Chin, J. P., McGrath, J. W., and Quinn, J. P. (2016) Microbial transformations in phosphonate
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(6) Horsman, G. P., and Zechel, D. L. (2017) Phosphonate Biochemistry. Chem. Rev. 117, 5704–
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L. (2012) PhnY and PhnZ comprise a new oxidative pathway for enzymatic cleavage of a
carbon-phosphorus bond. J. Am. Chem. Soc. 134, 8364–8367.
(8) Martinez, A., Tyson, G. W., and DeLong, E. F. (2010) Widespread known and novel
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