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Cytochrome P460 Cofactor Maturation Proceeds via Peroxide-

Dependent Post-translational Modification
Melissa M. Bollmeyer, Rachael E. Coleman, Sean H. Majer, Silas D. Ferrao, and Kyle M. Lancaster*
Cite This: J. Am. Chem. Soc. 2023, 145, 14404−14416 Read Online

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ABSTRACT: Cytochrome P460s are heme enzymes that oxidize hydroxylamine to

nitrous oxide. They bear specialized “heme P460” cofactors that are cross-linked to
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their host polypeptides by a post-translationally modified lysine residue. Wild-type N.

europaea cytochrome P460 may be isolated as a cross-link-deficient proenzyme
following anaerobic overexpression in E. coli. When treated with peroxide, this
proenzyme undergoes maturation to active enzyme with spectroscopic and catalytic
properties that match wild-type cyt P460. This maturation reactivity requires no
chaperones�it is intrinsic to the protein. This behavior extends to the broader
cytochrome c′β superfamily. Accumulated data reveal key contributions from the
secondary coordination sphere that enable selective, complete maturation.
Spectroscopic data support the intermediacy of a ferryl species along the maturation pathway.

■ INTRODUCTION
Post-translational modifications (PTMs)�covalent modifica-
tion of amino acids following protein translation�are an
important means by which nature extends the vast proteomic
complexity available through assemblies of the canonical amino
acids, cofactors, and prosthetic groups.1,2 Elucidation of both
the functional roles and origins of PTMs is a critical goal for
understanding natural biological systems. Moreover, insights
from such studies should afford valuable strategies for the
design of non-natural enzymes with tailored functions. One
class of PTM that has received substantial investigation are
heme-protein cross-links, which are ubiquitous and are
encountered in both eukaryotes and prokaryotes.3 Key Figure 1. Known heme-protein cross-links. The purple arrows
examples of these cross-links occur in c-heme proteins, where represent ester bonds to the heme methyl groups, the blue arrows
represent cross-links to the vinyl group of the heme, the red arrows
cysteine residues are attached to heme vinyl groups to form
represent the tyrosine-heme cross-link in HAO, and the green arrow
covalent thioether linkages (Figure 1).4 Additional, non- represents the lysine-heme cross-link in cyt P460. Adapted with
canonical cross-links are known, and relatively current permission from ref 3. Copyright 2015, Elsevier.
understanding of the origins and roles of these cross-links
has been reviewed by Lin.3 double cross-link to the meso-carbon and β-pyrollic positions of
Of the known, natural heme-protein cross-link PTMs, the c-heme (Figure 2). The presence of the cross-link alters the
almost all comprise covalent attachments to the heme electronics of this cofactor, giving rise to the characteristic 460
substituents�specifically the β-pyrollic methyl and vinyl nm Soret absorption maximum that it displays in its FeII form.
groups. Deviations from this trend, where the cross-link HAOs featuring heme-Tyr cross-linked cofactors oxidize
involves the porphyrin macrocycle itself, are thus far unique to NH2OH to nitric oxide (NO). HAO variants that do not
the heme P460 cofactors employed in the primary metabolisms possess the heme-Tyr cross-link are ineffective at NH2OH
of nitrifying and anaerobic ammonia oxidizing (anammox)
bacteria for hydroxylamine (NH2OH) redox catalysis.5 Heme
P460 proteins comprise hydroxylamine oxidoreductases Received: April 6, 2023
(HAOs) and cytochromes (cyts) P460. HAOs are soluble, Published: June 20, 2023
homotrimeric assemblies of octaheme subunits that support
one heme P460 catalytic center per monomer. These sites are
differentiated from the other cofactors by an open coordina-
tion site at the Fe and the presence (or absence) of a heme-Tyr

© 2023 American Chemical Society https://doi.org/10.1021/jacs.3c03608

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the highly ruffled nature of the P460 cofactor. If one assumes a

fully symmetric, planar heme cofactor to have D4h symmetry,
ruffling is a B1u distortion away from planarity.15 This type of
distortion of the macrocycle away from planarity has been
investigated at length and has been implicated in tuning heme
FeII/III reduction potentials and promoting directed, oxidative
heme degradation.16,17 Importantly, the aforementioned CLD
structure shows that ruffling in cyt P460 is not a consequence
of the cross-link but persists even without the cross-link.12
Given that cyt P460s are produced by obligate aerobic
organisms and that the cross-link forms even with recombinant
expression, we hypothesized that the heme-Lys cross-link
Figure 2. Heme P460 active site in N. europaea HAO (PDB 4N4N) forms in an oxygen-dependent process�similar to other non-
and cyt P460 (PDB 2JE3). Both are c-type hemes that feature canonical cross-links.18 To this end, we now report key
additional covalent attachments. In HAO, a tyrosine residue forms discoveries concerning the origin of the heme-Lys cross-link in
cross-links from the phenolate O to the pyrrole α-carbon and from Cε
to the α-meso-carbon. The heme in cyt P460 contains a cross-link cyt P460. We have identified conditions for expression and
from a lysine N to the γ-meso-carbon. purification of CLD proenzyme as well as for maturation of
this material into functional, cross-linked cyt P460. The
emergent picture exhibits considerable parallels to the
oxidation.6,7 Rather, they are proposed to serve as catalysts for reactivity of heme oxygenases,19 although with a key
reduction of nitrite (NO2−).8 Thus, the heme-Tyr cross-links divergence in outcome�productive enzyme maturation, rather
in HAO are proposed to bias HAOs toward oxidative than cofactor destruction.
chemistry. However, the ability to test this hypothesis is
limited by the lack of a viable recombinant expression platform
for oxidative HAOs, which precludes site-directed mutagenesis
■ MATERIALS AND METHODS
Anaerobic Expression and Purification of N. europaea cyt
experiments. Meanwhile, no successful attempts at installing P460. The pET22b(+) vector containing the gene for N. europaea cyt
cross-links in recombinantly expressed “reductive” HAOs have P460 was co-transformed with a pEC86 vector encoding for cyt c
been reported. maturation genes ccmABCDEFGH into E. coli strain BL21(DE3) and
Cyt P460 enzymes, however, can be recombinantly ex- selected for on lysogeny broth plates with 100 μg mL−1 ampicillin and
pressed.9,10 These soluble, homodimeric enzymes commonly chloramphenicol as previously described.10,20 A single colony was
feature a single heme P460 center per subunit whose γ-meso- used to inoculate a 5 mL lysogeny broth (LB) starter culture
containing 100 μg mL−1 ampicillin and 37 μg mL−1 chloramphenicol.
carbon is bound to the amine-terminated sidechain of a nearby After growing the starter culture overnight at 30 °C and 180 rpm, it
Lys residue. These cofactors also exhibit a 460 nm Soret was spun down and the cell pellet was brought into an anaerobic
absorption maximum in their reduced form. Cyt P460s have glovebox. Four septum-sealed, 1 L Schott bottles containing 750 mL
been shown to carry out the oxidation of two equivalents of of Terrific Broth (TB) medium supplemented with 0.5% glycerol,
NH2OH to generate nitrous oxide (N2O).10 We have ampicillin, and chloramphenicol were degassed by 2 cycles of
previously speculated that cyt P460 may play a role in evacuation for 12 min followed by sparging with N2 for 12 min.
preventing accumulation of cytotoxic cellular quantities of The media were brought into the glovebox and used to resuspend the
NH2OH and NO.10 Substitution of Lys with Tyr11 or Leu12 cell pellet. The suspension was used to inoculate the four bottles of
via site-directed mutagenesis generates c-type hemoproteins degassed media. Cultures were brought outside the glovebox and were
grown overnight at 37 °C and 60 rpm in a floor shaker. They were
that only contain the standard Cys cross-links. These variants induced with 0.4 M isopropyl β-D-1-thiogalactopyranoside (IPTG) at
are catalytically inactive toward NH2OH: They can form all 30 °C and 60 rpm for 8 h. Alternatively, the starter culture was used
intermediates of the NH2OH oxidation catalytic cycle via to inoculate two 6 L flasks containing 2 L of TB medium with
shunts, but the initial NH2OH oxidation events do not occur.11 ampicillin and chloramphenicol aerobically and grown at 30 °C
Complementary investigations of cyt P460 variants with overnight. Cells were pelleted at an OD600 between 0.7 and 1.0 to
substituted residues in their distal pockets combined with a prevent auto-induction. The brown cell pellet was brought into the
2.25 Å structure of a cross-link-deficient (CLD) cyt P460 box and distributed between four bottles of degassed TB medium
variant strongly suggest that the role of the heme-Lys cross-link prepared as described above. The cultures were induced with 0.4 M
is to position the cofactor sufficiently close to the carboxylate IPTG at 30 °C and 60 rpm for 8 h.
Cells were harvested by centrifugation of bottles loaded and sealed
group of a distal glutamate to enable proton transfer during the in the glovebox. Centrifugation was carried out at 4000g for 25 min.
initial step(s) of NH2OH oxidation.12,13 Pellets were returned to the glovebox, and the cells were then
While a clear picture is emerging of the functional role of the resuspended in 10 mL of degassed buffer containing 20 mM MOPS
heme-Lys cross-link, its origins have remained elusive. The (pH 8.0), 300 mM NaCl, 0.1% Triton X-100, and 2 mM EDTA. To
apparent non-necessity of molecular chaperones apart from lyse the cells, they were treated with 20 mg of lysozyme, 10 mg of
standard c-heme maturation proteins to successfully express DNase I, and 10 mg of RNase and then added to a Hungate tube that
functional, cross-linked cyt P460 has led to speculation that the was subsequently crimp sealed. These tubes were removed from the
heme-Lys PTM forms via an autocatalytic process.9 To date, glovebox and were incubated at 37 °C in a water bath for 1 h. The
no further progress had been reported. However, an early tubes were then returned to the glovebox, and the lysate was then
centrifuged in the glovebox in Eppendorf tubes at 14000 rpm for 60
crystal structure of Nitrosomonas europaea cyt P460 provides min. The supernatant was then diluted in a buffer containing 20 mM
some clues. This structure featured a second modification to MOPS (pH 8.0), 20 mM imidazole, and 150 mM NaCl. This solution
the c-heme: It is hydroxylated at its α-meso carbon, opposite to was applied to HisPur Ni-NTA resin (Thermo Scientific) packed in a
the heme-Lys cross-link on the γ-meso-carbon.14 Such gravity column. Once bound to the column, the protein was washed
susceptibility to oxidative damage is not unexpected due to with 40 mL of the aforementioned buffer and then was eluted with a

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Figure 3. Hypothetical mechanism of peroxide-dependent heme-Lys cross-link formation in cyt P460. (A) Possible routes for hydroxylation of the
meso-carbon followed by (B) condensation by lysine.

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Figure 4. Characterization of the anaerobically purified proenzyme and matured proenzyme, which was generated by reaction of the proenzyme
with 3 equivalents of Li2O2 followed by quenching with sodium dithionite, re-oxidization with [Ru(NH3)6]Cl3, and washing. (A) UV/vis
absorption spectra of the proenzyme (red) and matured enzyme (black). (B) CW X-band EPR spectra collected at 12 K of the proenzyme (red)
and matured enzyme (black). Gray traces are the corresponding simulations. (C) Resonance Raman spectra obtained via excitation at 405 nm for
the proenzyme (red), matured (black), and WT cyt P460 (green). (D) Specific activities for WT cyt P460, the proenzyme, and matured proenzyme
quantified by the 280 nm extinction coefficient (gray) or the 440 nm extinction coefficient (black). Error bars represent standard deviations of three
trials.

second buffer comprising 20 mM MOPS (pH 8.0) containing 150 protein was washed with 20 mM Tris/200 mM NaCl (pH 8.0). To re-
mM NaCl and 330 mM imidazole. The fractions containing the target oxidize the protein, [Ru(NH3)6]Cl3 was added and subsequently
protein were buffer exchanged into 20 mM Tris (pH 8.0) using removed by buffer exchange. The Arg44Ala mutant was matured in a
centrifugal filtration columns. The resulting protein solution was then similar fashion, but instead using 8−12 equivalents of Li2O2. To
applied to a Q Sepharose Fast Flow (GE Healthcare) gravity column. determine Li2O2 concentration dependence of the maturation
The protein on the column was washed with 3 column volumes of 20 reaction, the same procedure was followed maintaining proenzyme
mM Tris (pH 8.0) followed by 3 column volumes of 20 mM Tris (pH at 8 μM and adding 3, 6, or 9 equivalents of Li2O2.
8.0) with 100 mM NaCl. The protein was eluted with 20 mM Tris Maturation of the proenzyme using glucose oxidase (GOD) to
(pH 8.0) containing 200 mM NaCl yielding homogeneously pure cyt generate O22− was carried out by first adding 12 μM cyt P460 to an
P460. Purity was assessed by SDS-PAGE (Figure S1). Proenzyme anaerobic cuvette containing 20 mM Tris/200 mM NaCl (pH 8.0)
concentrations were quantified using the extinction coefficient at 280 such that the final volume would be 2 mL. The cuvette was
nm for WT cyt P460 (ε280 = 38.9 mM−1cm−1). equilibrated at 25 °C with stirring for at least 3 min. Full-wavelength
Maturation Experiments. To mature cyt P460 proenzyme using scans were then collected every 0.3 min. After 3 min, 50 μL of 1 mg/
oxygen, a final concentration of 8 μM proenzyme was added to an mL GOD was added to the cuvette using a Hamilton syringe. After
anaerobic Spectrosil quartz cuvette (Starna Cells, 10 mm) containing another 3 min, the cuvette was opened to the atmosphere. The
a final volume of 2 mL in 20 mM Tris/200 mM NaCl (pH 8.0). The reaction was initiated after 10 min of equilibration with the
cuvette was equilibrated in the UV/vis spectrometer at 25 °C with atmosphere by addition of 500 μM glucose.
stirring for at least 3 min and was then opened to the atmosphere. In For Li2O2 reactions in the presence of guaiacol, 2 mM guaiacol was
cases where O2 was added, a needle was inserted into the cuvette and added to the anaerobic cuvette with the protein solution and was
O2 was gently passed through the protein solution. Scans from 200− equilibrated at 25 °C with stirring for at least 3 min. Scans were
800 nm were recorded every 10 min during experiments under air or recorded every 0.2 min. The reactions were initiated by addition of
every 0.3 min when O2 was bubbled. Li2O2 via a Hamilton syringe.
For cross-link reactions using Li2O2, an anaerobic cuvette
containing 8 μM proenzyme in 20 mM Tris/200 mM NaCl (pH
8.0) was equilibrated at 25 °C with stirring for at least 3 min. Full-
wavelength scans were recorded every 0.3 min for several minutes
■ RESULTS AND DISCUSSION
Analogy to Heme Oxygenase Reactivity. Two key
before the addition of 3 equivalents of Li2O2. Scans were recorded observations led us to hypothesize that modification of the
until there were no longer changes in the UV/vis spectra, at which heme P460 γ-meso-carbon proceeds via a reaction similar to
point excess sodium dithionite (Na2S2O4) was added to quench the those reported for heme oxygenases.17,21 One is the
reaction. The cuvette was returned to the glovebox, after which observation by Wilmot and co-workers of hydroxylation at
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the cyt P460 heme α-meso-carbon that presumably arose from Table 1. Resonance Raman Shifts of Porphyrin Marker
oxidative damage. The second is the high degree of ruffling Bands from N. europaea cyt P460 Variantsa
common to both heme P460 cofactors and hemes bound by
ν3 ν4 ν10
heme oxygenases. cyt P460 variant (cm−1) (cm−1) (cm−1) ref
Heme oxygenase chemistry is principally driven by reactive
Lys70Tyr cyt P460 (CLD) 1501 1372 NR 11
oxygen species.22 One outcome of heme oxygenation is meso-
WT cyt P460, aerobic 1504 1359 1614 this work
carbon hydroxylation, although the mechanism by which this preparation
proceeds remains a topic of investigation.22−24 In brief, WT cyt P460 proenzyme 1488 1369 1616 this work
possibilities include formation of a FeIII-heme peroxo or (CLD)
hydroperoxo intermediate that directly attacks the meso- WT cyt P460 matured enzyme 1504 1364 1615 this work
carbon, or this intermediate could undergo homolytic cleavage Arg44Ala (CLD) 1487 1367 1618 this work
to form ferryl compound II (FeIV�O) and a transient a
Obtained using 405 nm laser excitation.
hydroxyl radical. In non-canonical heme oxygenases such as
IsdG, a significant ruffling deformation of the heme was shown EPR spectrum obtained at 12 K revealed a mixture of S = 5/2
to allow for selective hydroxylation of the β- or δ-meso- signals that were consistent with both mature and CLD cyt
carbons.25 The substantial, cross-link-independent ruffling of P460 (Table 2). The CLD signal was the major component,
the cyt P460 macrocycle could allow for a similar mechanism comprising 80% of the mixture, while mature enzyme
to hydroxylate the γ-meso-carbon, which would conveniently constituted the remaining 20%.
supply a functional group that can undergo keto−enol
tautomerization. Although heme oxygenases have not been Table 2. Spin Hamiltonian Parameters Obtained from
shown to selectively hydroxylate a γ-meso-carbon, differences in Fitting X-Band EPR Spectra of cyt P460 Variants
the active site of cyt P460 may allow for control over this
regioselectivity. The keto form will be susceptible to species (FeIII, S = 5/2)
condensation with the proximal Lys amine, thus forming the cyt P460 variant gef f E/D ref
C−N cross-link and the loss of a water molecule. Alternatively, Lys70Tyr cyt P460 (CLD) 5.78, 1.98 0.00 11
the mechanism could be similar to peroxidases where a heme WT cyt P460, aerobic preparation 6.57, 5.09, 0.03 10
peroxo species undergoes heterolytic cleavage to compound I 1.97
(FeIV�O porphyrin radical cation), which could participate in WT cyt P460 proenzyme, minor 6.52, 5.06, 0.03 this
component(CL) 1.97 work
oxygen atom transfer reactions with the meso-C. However,
WT cyt P460 proenzyme, major 6.02, 5.54, 0.01 this
compound I of heme oxygenase was shown to be incompetent component (CLD) 1.99 work
for heme hydroxylation.26 These possible mechanisms are WT cyt P460 matured enzyme 6.50, 5.06, 0.02 this
summarized in Figure 3. 1.97 work
Anaerobic Overexpression Yields Cross-Link-Defi- Arg44Ala (CLD) 5.69, 1.99 0.00 this
cient cyt P460 Proenzyme. Given the dependence of work
heme oxygenase chemistry on reactive oxygen species, we Arg44Ala matured, major component 5.76, 1.99 0.00 this
work
leveraged the facultative anaerobic nature of E. coli to carry out
Arg44Ala matured, minor component 5.38, 2.00 0.00 this
anaerobic overexpression and purification of wild-type (WT) work
N. europaea cyt P460. Gratifyingly, expression of the protein NpAv Leu105Lys 6.63, 5.07, 0.02 this
under these conditions led to the isolation of a red protein with 1.96 work
spectroscopic features characteristic of cross-link-deficient
(CLD) cyt P460, although with yields significantly lower Cyt P460 Maturation Is Driven by Peroxide. Given that
than those obtained following aerobic expression. Produced the formation of the cross-link appeared dependent on oxygen
this way, WT N. europaea cyt P460 exhibits a Soret maximum during expression, anaerobically purified proenzyme was
at 404 nm (Figure 4). The presence of a shoulder at 440 nm exposed to ambient air to determine whether O2 is sufficient
was consistent with a minor amount of cross-linked protein to drive enzyme maturation. Upon opening an anaerobic
(vide infra). This presumably formed from small amounts of cuvette containing proenzyme to air, the 404 nm proenzyme
oxygen present during protein overexpression. Soret absorption decayed slowly, and a new feature at 413 nm
A resonance Raman (rR) measurement using 405 nm laser grew in intensity. The appearance of the 413 nm feature
excitation yielded a spectrum consistent with a ruffled c-heme, coincided with an increase in absorbance at 440 nm assigned
as encountered in previously reported CLD cyt P460 variants to matured cyt P460 (Figure S2). After 16 h, the reaction
(Table 1). The oxidation state marker band (ν4) occurs at remained incomplete. After 2 days, the heme absorbance was
1369 cm−1, consistent with a ferric assignment for the heme. almost entirely diminished, consistent with cofactor degrada-
The spin state marker band (ν3) occurs at 1488 cm−1.27 While tion rather than maturation. In summary, exposing the
the energy of the ν3 band is lower than that reported for the proenzyme to O2 in air led to only a slow (kobs(440) = 0.0018
cross-link-deficient N. europaea Lys70Tyr at 1501 cm−1, it is min−1) conversion to cross-linked enzyme and, ultimately,
consistent with energies expected for high-spin ferric hemes.11 heme degradation.
The ν10 band, known to be sensitive to out-of-plane distortions We next considered the participation of reactive oxygen
of the heme, was assigned to the peak at 1616 cm−1 based on species in driving cofactor maturation. To this end, we treated
previous assignments for c-type hemes.28,29 In WT cyt P460, the proenzyme with 3 equivalents of lithium peroxide (Li2O2).
this band appears at 1614 cm−1. These similar frequencies Initial attempts using H2O2 yielded inconsistent results, likely
suggest that the CLD proenzyme remains ruffled, in accord owing to the difficulty in quantifying H2O2 and the sensitivity
with the structural data obtained for CLD Lys106Leu/ of the protein to degradation if excess peroxide is added. Using
Ala131Glu N. sp. AL212 cyt P460.12 A spin-counted X-band Li2O2 instead allowed for better control over the concentration
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by dissolving a known amount in buffer. Regardless, 5.68, 2.00], a small S = 1/2 component with g = [2.01, 2.02,
considering the pKa of H2O2 is 11.6, dissolution of Li2O2 in 2.01], and a third feature at g = 4.28 (Figure S5). The new S =
a pH 8 buffer would generate H2O2. 5/2 species can plausibly be assigned to a hydroxylated heme
The previously observed 413 nm intermediate formed that would eventually proceed toward degradation. The S = 1/
immediately after addition of 21 μM Li2O2 to 7 μM proenzyme 2, meanwhile, is not consistent with that of any characterized
by manual mixing (Figure 5). This intermediate subsequently compound I species. The reaction of proenzyme with 3
equivalents of Li2O2 frozen after 4 min contained the same
three species in its EPR spectrum. To further characterize the
product of the reaction of WT cyt P460 with Li2O2, an 57Fe
Mössbauer spectrum was obtained for 57Fe-enriched WT
protein that had been treated with 3 equivalents of Li2O2 and
frozen after 4 min (Figure S6). This spectrum contained two
Fe-containing components. The minor component, accounting
for 29% of the signal, had an isomer shift (δ) of 0.59 mm/s and
quadrupole splitting of 0.72 mm/s, which is consistent with the
new S = 5/2 signal observed in the EPR spectrum. The major
component had an isomer shift of 0.09 mm/s and quadrupole
splitting of 1.60 mm/s, which are characteristic of FeIV�O-
containing species such as compound I or compound II.30
Given that this species was EPR-silent, it can plausibly be
assigned as compound II.
Matured Protein Matches Properties of the Aerobi-
Figure 5. Reaction of 7 μM proenzyme (red) with 3 equivalents (21 cally Expressed cyt P460. To further characterize the
μM) of Li2O2 at 25 °C. Scans were recorded every 0.3 min. The scan protein resulting from the maturation of proenzyme with
immediately following Li2O2 addition is shown in black. The final peroxide, we obtained an X-band EPR spectrum at 12 K of
scan is shown in blue, after which the reaction was quenched with
Na2S2O4. Time courses of the absorbances corresponding to the
matured protein that was treated with Na2S2O4 and re-oxidized
proenzyme (404 nm) and product (460 nm) are shown in the inset. (vide supra). The signal corresponding to the proenzyme was
completely absent. The only signal present was that of a
rhombic S = 5/2 system with g values of 6.50, 5.06, and 1.97
decayed, yielding a final spectrum with a broad Soret (E/D = 0.02). These values are consistent with those of native,
absorption with a maximum near 445 nm. After ca. 4 min, cross-linked WT cyt P460 (Table 2). Additionally, the rR
there were no further spectral changes. The reaction was then spectrum obtained via excitation at 405 nm exhibited an
quenched by addition of Na2S2O4. UV/vis absorption increased number of bands in accord with the descent in
spectroscopy of the resulting solution exhibited the 460 nm cofactor symmetry attending cross-link formation (Figure 4).
Soret band characteristic of FeII cyt P460. Following removal This spectrum of Li2O2-matured cyt P460 is indistinguishable
of Na2S2O4, the protein was treated with the oxidant from WT cyt P460 purified from aerobic growths.
[Ru(NH3)6]Cl3. The resulting UV/vis absorption spectrum To establish whether in vitro maturation imbues cyt P460
was identical to that of WT FeIII cyt P460 (Figure 4A). As an with NH2OH-oxidation activity, we carried out steady-state
alternative to adding Li2O2, peroxide could be generated within activity assays (Figure 4). Under anaerobic conditions, 2 mM
the reaction mixture using GOD, which oxidizes glucose to NH2OH was added to a cuvette containing 50 μM DCPIP and
H2O2 and D-glucono-δ-lactone. Addition of 500 μM glucose to 6 μM PMS followed by addition of 1 μM protein. The specific
a cuvette containing 12 μM proenzyme and 156 nM GOD activity obtained by monitoring consumption of DCPIP for the
resulted in formation of the 413 nm intermediate, which proenzyme was 4.5 ± 0.7 μM DCPIP μM cyt P460−1 min−1,
decayed to the same broad spectrum observed for the reaction significantly less than that of WT cyt P460 (20 ± 3 μM DCPIP
with Li2O2 (Figure S3). μM cyt P460−1 min−1). This lowered activity of the proenzyme
The final spectrum obtained following the maturation was expected due to the predominantly CLD character of
reaction before quenching with Na2S2O4 differs from matured proenzyme preparations. We attributed the minimal activity
WT P460. We speculated that this could occur for one of two observed to the presence of a minor amount of cross-linked
reasons. One is that the matured P460 subsequently reacts protein (vide supra). After maturing the proenzyme with Li2O2
with the remaining excess peroxide. Alternatively, the observed and removing all reagents by washing in a spin column, the
species could be the product following activation of peroxide specific activity was restored to 18 ± 1 μM DCPIP μM cyt
to modify the macrocycle, e.g., a ferryl species such as P460−1 min−1 with protein quantified using the heme Soret
compound I or II. These high-valent FeIV-oxo species are used maximum. This value is within the error of values obtained
in the catalytic cycles of several heme enzymes including cyt previously for WT cyt P460. There was, however, some
P450 and peroxidase. Compound I refers to an FeIV-oxo heme evidence of heme loss by the protein, as quantification on the
with a porphyrin radical cation, which can be reduced by one basis of total protein yielded a specific activity of 12.4 ± 0.8
electron to form compound II. To test whether CL cyt P460 μM DCPIP μM cyt P460−1 min−1. While we do not observe
could react with peroxide to form a ferryl species, 4 μM formation of a peptide-linked biliverdin, we cannot rule it out
aerobically grown, fully mature WT cyt P460 was allowed to on the basis of UV/vis and would need HPLC and MS analysis
react with 3 equivalents (12 μM) of Li2O2. The 440 nm Soret of the products, which is beyond the scope of this work.31
evolved to the same final spectrum observed during the Nevertheless, the restoration of WT cyt P460 spectroscopic
proenzyme maturation (Figure S4). X-band EPR at 10 K for properties as well as NH2OH oxidation activity to inactive
this species contained a new S = 5/2 feature with g = [6.25, proenzyme produced during anaerobic expression strongly
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evidences the peroxide dependence of cyt P460 cofactor

maturation.
Identity of the 413 nm Intermediate. A key common
observation from the maturation experiments described above
is that of the 413 nm intermediate. This intermediate species
was observed in reactions employing either air (O2) or
peroxide for maturation. It is the only intermediate observed
by UV/vis during the maturation reaction, and it is
isosbestically converted to the final product. Thus, its
consumption is the rate-determining step of the reaction.
Plausible identities for the 413 nm intermediate are [Fe−O2]1+
(e.g., FeIII−O22− or FeII−O2), FeIII−OOH−, compound I, or
compound II. Treatment of the proenzyme with meta-
chloroperoxybenzoic acid (m-CPBA) showed no reactivity
(Figure S7), suggesting that compound I is not formed by the Figure 6. Continuous-wave X-band EPR spectrum collected at 10 K
proenzyme. of the rapid freeze-quench reaction of 200 μM proenzyme with 400
Stopped-flow UV/vis absorption spectroscopy was carried μM Li2O2 frozen at 1 s (black) with the simulated spectrum (red).
out to scrutinize the reactivity of the 413 nm intermediate on The fit gave g values of 2.08, 2.01, and 1.98. Oxygen background was
the ms−s timescale. Rapidly mixing 10 μM CLD protein with removed from the raw experimental spectrum and the cavity was
2 equivalents of Li2O2 (20 μM) and recording spectra every 8 subtracted.
ms revealed a decay of the 404 nm peak and shift to 413 nm
within 2 s (Figure S8). A linear correlation between the guaiacol to tetraguaiacol, which exhibits a characteristic
observed rate of the 404 nm decay (after addition of Li2O2) absorption at 470 nm.35 The high-valent ferryl species
and Li2O2 concentration suggests that the kobs for the compound I and compound II are strong oxidants that are
conversion of CLD cyt P460 to the 413 nm intermediate able to activate relatively inert bonds. Peroxidases use these
exhibits a first-order dependence on [Li2O2], with a rate ferryl intermediates formed from peroxide to oxidize a variety
constant of 0.13 s−1 μM−1 (Figure S9). Meanwhile, the rate of of substrates, including guaiacol. Guaiacol oxidation has thus
decay of the 413 nm intermediate to mature enzyme exhibits been a marker of peroxidase activity, where the oxidation
no dependence on [Li2O2] (Figure S10). This 413 nm power of compound I or compound II is required. When Li2O2
intermediate ultimately leads to the final species that features a was added to the proenzyme with excess (2 mM) guaiacol,
broad Soret and Q-bands. there was an immediate formation of a broad absorbance at
The 413 nm species was trapped by RFQ of the reaction of 470 nm (Figure 7). The Soret band of the proenzyme did not
200 μM proenzyme with 400 μM Li2O2 at 0.5 s and 1 s time change, and after 30 min, the mixture was reduced to reveal
points. Based on the UV/vis absorption time course of this completely unreacted proenzyme. The peak at 460 nm is
reaction, the 413 nm intermediate should be mostly formed at attributed to cross-linked protein in the as-isolated sample that
0.5 s and completely formed after 1 s. The EPR spectra for formed as a result of some oxygen present during growth.
both the 0.5 s and 1 s sample are absent of any S = 5/2 signals Oxidation of guaiacol thus intercepted the intermediate
and show formation of a new S = 1/2 species (Figure S11). responsible for cross-link formation. While concerted hydrox-
The absence of a high-spin feature strongly suggests that the ylation of the meso-carbon would not be affected by the
413 nm intermediate is not the meso-hydroxylated heme but presence of guaiacol, a compound I or compound II
rather is an Fe-bound peroxo or superoxo adduct.32 Simulation intermediate would be competent for guaiacol oxidation.26,36
of the low-spin signal gave g values of 2.08, 2.01, and 1.98 A non-canonical heme oxygenase IsdI that hydroxylates heme
(Figure 6). Interestingly the g value of 2.08 is lower than those via a concerted mechanism was shown to proceed even in the
typically observed for ferric-peroxo or hydroperoxo inter- presence of guaiacol and did not result in guaiacol oxidation,
mediates, which have a higher g1 and larger spread of g values while generation of the ferryl species by the O-atom donor m-
(g1 > g2 > ge > g3).33,34 For example, a ferric-hydroperoxo CPBA was competent for guaiacol oxidation.37 In the case of
intermediate of heme-oxygenase was reported to have g values cyt P460, the cross-link mechanism does not proceed in the
of 2.37, 2.187, and 1.924.19 Instead, the EPR spectrum of the presence of guaiacol and instead only guaiacol oxidation is
413 nm intermediate is more consistent with a ferrous- observed, indicating that a ferryl species is responsible for
superoxo species, which are characterized by g1 ≫ g2 ≳ g3 ≈ ge. cross-link formation.
A key example of such a species is cryoreduced oxy- Both the low-signal EPR spectrum and oxidation of guaiacol
hemoglobin, which exhibits g1 = 2.11 and g2,3 ≈ ge.33 It is suggest maturation of the proenzyme involves a step that forms
unlikely, however, that the observed EPR spectrum corre- a ferryl intermediate. The sharp UV/vis Soret band and
sponds to the 413 nm intermediate. Given the low intensity of absence of a radical in the EPR spectra are consistent with this
the signal, we conclude that this is a minor species. Another intermediate being a compound II species, whereas compound
EPR silent species is likely present that could be FeII or FeIV. I typically has broad UV/vis features and a porphyrin radical
Thus, the EPR plausibly corresponds to minor accumulation of signal in the EPR spectrum.30 Fe K-edge X-ray absorption
a precursor to the 413 nm intermediate, which is itself silent spectroscopy of the intermediate, trapped by reaction of 500
under our EPR conditions. μM proenzyme with 500 μM Li2O2 after 1 s, showed a shift in
To obtain further support for ferryl formation during the pre-edge to 7114.2 eV compared to that of WT at 7112.4
maturation, the proenzyme was allowed to react with Li2O2 eV consistent with oxidation to a formally FeIV center (Figure
in the presence of guaiacol. Guaiacol is commonly used in 8). The pre-edge was also more intense, which has been
peroxidase assays, during which heme ferryl species oxidize observed for other compound II species and is consistent with
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Figure 7. (A) UV/vis spectral time course for the reaction of the proenzyme with 8 equivalents of Li2O2 in the presence of excess (2 mM) guaiacol.
The red trace is before Li2O2 was added, the black trace was immediately after addition, and the blue trace was 30 min after addition. Gray traces
represent scans every 0.2 min. (B) UV/vis spectrum from the reaction after addition of Na2S2O2.

unprotonated FeIV−oxo. Typically compound II species

captured for EXAFS exhibit distances closer to 1.65 Å that
are consistent with the unprotonated Fe(IV) −oxo.38,39
The data in sum are consistent with formation of compound
II. However, more work will be needed to clarify the
protonation state. Homolytic cleavage to compound II would
produce a hydroxyl radical that could hydroxylate the γ-meso-
carbon, allowing Lys to condense and form the cross-link.
Outer coordination sphere effects likely control the regiose-
lectivity of such a reactive radical, which would otherwise
indiscriminately attack the porphyrin. The mechanism in cyt
P460 may be similar to that of heme oxygenases, where a distal
water cluster H-bonded with an Arg-Asp pair was suggested to
play an important role in both stabilizing a hydroxyl radical and
Figure 8. Fe K-edge XAS XANES region for WT cyt P460 (green) directing it toward the α-meso-carbon.40 Tautomerization of a
and the reaction of ca. 500 μM proenzyme with 1 equivalent of Li2O2 hydroxylated meso-carbon would then provide an electrophilic
frozen at 1 second (black). carboxyl group for the nucleophilic lysine to attack and form a
covalent attachment. The nucleophilic nature of the lysine
sidechain allows for a large diversity of covalent modifications
a descent in symmetry at the Fe center promoting 3d/4p including methylation, acetylation, carbonylation, among
mixing. Fitting of the extended X-ray absorption fine structure others.41,42 Carbonylation has been shown to correlate with
(EXAFS) region (Figure S12) over a k-range of 2−13 Å oxidative stress, and recently lysine carbonylation was observed
revealed an Fe−O scatter with a distance of 1.83 Å (Table S2). in cyt c induced by reactivity with peroxide.43 While many
This distance is consistent with a protonated FeIV−OH PTMs require additional enzymes, both cyt c and cyt P460
species, i.e., protonated compound II. However, the increased catalyze their own active-site modifications using peroxide as a
intensity of the pre-edge is more consistent with an substrate, allowing them to gain new functions.

Figure 9. (A) Structure of WT N. europaea cyt P460 (PDB 2JE3) (B) Structure of the N. europaea cyt P460 Arg44Ala mutant (PDB 8GAR). (C)
Overlay of the heme groups from the WT (green) and Arg44Ala (gray) cyt P460 structures, highlighting the change in position of the 6-β-pyrrolic
propionate and γ-meso-carbon.

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Influence of Hydrogen Bonding Networks on Table 3. Data Collection and Refinement Statistics for N.
Cofactor Maturation. Heme ruffling is known to control europaea Arg44Ala cyt P460
function, like that of nitrophorins44,45 or heme oxygenase.25
wavelength (Å) 0.979
Cytochrome P460 shares in this ruffling deformation even in
temperature (K) 80
cross-link-deficient variants. If the Lys is not responsible for
space Group P 31 2 1
ruffling, there must be other structural factors that are
a (Å) 53.111
responsible. Inspection of the N. europaea cyt P460 active
b (Å) 53.111
site reveals a distal Arg residue, Arg44, that is involved in a
c (Å) 125.894
hydrogen-bonding (H-bonding) network that engages the
α (deg) 90
heme 6-β-pyrrolic propionate (Figure 9). We suspected that
β (deg) 90
this H-bond network contributes to ruffling the heme P460
γ (deg) 120
cofactor. To test whether this Arg is indeed promoting ruffling
no. of reflections 606,675 (60,977)
and by extension to test whether the ruffling deformation is
no. of reflections in the Rwork set 30,604 (2,999)
required for cross-link formation, we generated the Arg44Ala
no. of reflections in the Rfree set 1454 (150)
cyt P460 mutant. Purification of this mutant yielded a resolution (Å) 43.2−1.55 (1.605−1.55)
completely CLD protein despite being grown under aerobic Rmerge (%) 0.05202 (1.518)
conditions (Figure S13A). The EPR spectrum obtained at the Rmeas (%) 0.05344 (1.557)
X-band and at 10 K was axial with g = [5.69, 1.99] (Figure CC1/2 1 (0.916)
S13B). The rR spectrum obtained via excitation at 405 nm completeness (%) 1.00 (1.00)
resembled that of the WT proenzyme (Figure S13C) with little redundancy 19.8 (20.2)
deviation in the porphyrin marker bands (Table 1). I/σ(I) 32.17 (2.11)
Having established that Arg44 influences cross-link for- Rwork 0.1635 (0.2603)
mation, we crystallized CLD Arg44Ala cyt P460 to probe the Rfree 0.1968 (0.3169)
role of the Arg44 H-bond in the active site structure. Red root-mean-square deviation from ideality
diamond-shaped crystals of N. europaea Arg44Ala cyt P460 bonds (Å) 0.009
were grown in 38−42% PEG 3000, 0.2 M calcium acetate angles (deg) 0.98
hydrate, and 0.1 M sodium cacodylate (pH 6.5) using the average B factor (Å2) 34.37
sitting drop method. The largest crystals were obtained using 9 Ramachandran plot
μL of 350 μM protein in 50 mM MOPS (pH 8.0) with 1 μL of favored regions (%) 97
crystallization buffer and grew in 5 days. These crystals allowed regions (%) 2.6
diffracted to 1.55 Å (Table 3). Density for an active-site disallowed regions (%) 0
capping loop region was absent in our data as it was with PDB ID 8GAR
Wilmot’s WT structure.14 Previously, this was attributed to
oxidative damage due to the protracted crystallization period.
Given our briefer crystallization period, this could instead be be fit with two axial, high-spin components. The first
due to this loop being highly flexible in the N. europaea protein. component accounted for 34% of the total spin and had g =
The impact of the Arg44Ala mutation on the heme [5.76, 1.99] while the second component accounted for 58% of
architecture was apparent: the 6-β-pyrrolic propionate that the spin and had g = [5.38, 2.00]. The rest of the spin was
interacts with Arg44 in WT cyt P460 was significantly accounted for by a g = 4.24 signal that was likely a result of
perturbed. The propionate’s carboxyl carbon shifts by 1.1 Å heme degradation. Maturation of the Arg44Ala mutant
compared to WT. Removing the Arg-propionate interaction restored hydroxylamine oxidation activity (Figure S13D),
effectively eliminated any heme ruffling (Figure 9). Normal giving strong evidence that the cross-link was formed.
coordinate structural decomposition showed that the ruffling However, the presence of a second species identified by the
deformation was only 0.1 Å, about 7-fold less than that of WT UV/vis and EPR spectra of the product suggests non-specific
(Figure S14). Notably, the γ-meso-carbon was shifted by 0.9 Å reactivity. Although the ruffling deformation was not required
relative to WT. While the ruffling deformation was significantly for reactivity with peroxide, it can explain the apparent
altered, the heme maintained its saddling deformation of 0.8 Å. unselective product formation. Heme ruffling is expected to
The cross-linking lysine residue�which has previously never localize spin density onto the meso-carbons, making them more
been observed in a crystal structure of cyt P460 without being susceptible to hydroxylation.46 Without this influence of
covalently attached to the porphyrin�formed a salt-bridge ruffling on the meso-carbons, in particular the γ-meso-carbon
with Glu96 in our structure. Prior to covalent attachment, the of cyt P460, the reactive intermediates such as the FeIII-peroxo
lysine sidechain is free to interact within the active site and or compound II could indiscriminately attack the porphyrin
could play an important outer-sphere role during cross-link ring leading to non-specific heme hydroxylation.
formation. Influence of Arg44 on the Mechanism of Cross-Link
Reaction of the as-purified Arg44Ala cyt P460 with 8 Formation. It is evident that Arg44 plays an important but
equivalents of Li2O2 resulted in formation of a 410 nm non-essential role in cross-link formation. To determine
intermediate that converted to a broad Soret maximum whether the mechanism was the same as the WT proenzyme,
centered near 438 nm (Figure S15). When the reaction was the reaction was studied by stopped-flow kinetics. Stopped-
quenched with Na2S2O4, the product featured a new Soret flow reactions with varying concentrations of Li2O2 showed
band at 457 nm that resembled FeII cross-linked cyt P460. formation of an intermediate with a Soret band at 410 nm and
After re-oxidation using [Ru(NH3)6]Cl3, the UV/vis spectrum Q-bands at 524 and 557 nm (Figure 10). This species decayed
had a broad Soret band at 432 nm and a new feature at 413 nm rapidly to the final product, in contrast to the WT proenzyme,
(Figure S13A). The EPR spectrum of this FeIII product could which did not convert to the final product on this time scale.
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Figure 10. (A) UV/vis spectral time course for the stopped-flow reaction of 10 μM Arg44Ala with 8 equivalents (80 μM) Li2O2. The red trace is
the first spectrum after mixing, and the blue trace is the final spectrum at 30 s. The inset shows the absorbance at 403 nm over time fit to a double
exponential. (B) UV/vis spectrum comparing as-isolated Arg44Ala (red) and the intermediate formed during the stopped-flow reaction (black).

Decay of the absorbance at 403 nm could not be fit to a single is highly conserved across the cytochrome P460 protein family.
exponential and was better fit by a double exponential. The A sequence logo generated from a multiple sequence alignment
two components of this fit correspond to the formation of the of 236 annotated cyt P460 species reveals that the
410 nm intermediate, which still absorbs at 403 nm, followed predominant residue at the cross-link position is Lys (Figure
by the decay of the intermediate to give the final species. S19), appearing in approximately 73% of the sequences. The
Plotting kobs of the first step against the concentration of Li2O2 next most frequent is Leu in about 10% of the sequences. The
ranging from 60−160 μM showed saturation (Figure S16). sequences lacking the Lys cross-link residue are better
The observed rate at 160 μM was 1.89 s−1, lower than that classified in the cyt c′β subfamily, which is closely related to
obtained for the WT proenzyme at only 30 μM Li2O2 (3.67 cyt P460.49 Cyt c′β proteins have been implicated in NO
s−1). The requirement for higher concentrations of Li2O2 to binding and are proposed to play a role in NO detoxification,
react indicates that the Arg44Ala mutant has a lower binding although their function in vivo remains unknown.50 Crystal
affinity for peroxide. The slower rate of formation of the structures of cyt c′β from both N. europaea and Methylococcus
intermediate is consistent with the Arg H-bond network capsulatus have shown that these proteins have the same
facilitating O−O bond cleavage. These data together suggest overall fold as cyt P460 and overlay well with the N. europaea
that Arg44 could play a role in interacting with the bound cyt P460 structure.51,52 In place of the cross-linking lysine,
peroxo ligand. In peroxidases, a distal Arg aids in O−O these proteins contain a methionine (Met79) or phenylalanine
cleavage by providing a H-bond, but it is not required for (Phe61), respectively, that do not form cross-links. We
cleavage to occur.47,48 identified a cyt c′β gene from Nitrosospira sp. NpAV that
Based on the stopped-flow kinetics, samples of 200 μM contains a Leu (Leu105) residue in the cross-linking position.
R44A mixed with 1600 μM Li2O2 were subjected to RFQ at To test whether the conserved protein fold is sufficient to
100 ms, 400 ms, 600 ms, and 4 s. Only the signal of the as- promote cross-link formation given the presence of an
isolated species was observed for the 100 and 400 ms samples, appropriate residue, we expressed and purified WT cyt c′β
and after 600 ms, there was no observable signal (Figure S17). from Nitrosospira sp. NpAv along with a Leu105Lys mutant.
This EPR-silent intermediate is consistent with the mechanism Purified WT Nitrosospira sp. NpAv cyt c′β is a red protein
of the WT proenzyme, which we posit proceeds through EPR- with a Soret maximum at 400 nm and shoulder at ca. 378 nm
silent compound II. To determine whether the compound II (Figure S20), which is consistent with cyt c′β from both N.
intermediate is responsible for cross-link formation in the europaea and Methylococcus capsulatus.51,53 When reacted with
Arg44Ala mutant, the reaction was repeated in the presence of Li2O2 at concentrations comparable to the cyt P460 reactions
guaiacol (Figure S18). Upon addition of Li2O2, the 470 nm above, Nitrosospira sp. NpAv cyt c′β showed no reactivity. This
peak of oxidized guaiacol formed immediately. The Soret band contrasts with cyt c′β from N. europaea, although the
of CLD Arg44Ala remained unchanged during the reaction. concentrations used in that study were 10-fold higher. It is
Reduction of the product with Na2S2O4 yielded a UV/vis possible that the residue in the cross-linking position plays a
lacking the 457 nm Soret band corresponding to cross-linked critical role in peroxide reactivity. Substitution of Lys in the
protein, in contrast to the reaction without guaiacol. Guaiacol cross-linking position yielded a green protein with a Soret
therefore intercepts compound II formation and prevents maximum at 442 nm. The overall shape of the UV/vis
cross-link formation. Compound II is responsible for cross-link absorption spectrum was similar in appearance to WT N.
formation in both the WT proenzyme and the Arg44Ala europaea cyt P460. When the Leu105Lys Nitrosospira sp. NpAv
mutant, but removal of the H-bond network supported by Arg cyt P460 variant is reduced, the Soret maximum shifts to 460
impacts the ability to selectively form the cross-link. Without nm. The X-band EPR spectrum of the Leu105Lys variant
the H-bond network, the reactivity of compound II cannot be collected at 12 K had a gmax, gmid, and gmin of 6.63, 5.07, and
tightly controlled for hydroxylation of the γ-meso-carbon, 1.96 with an E/D of 0.02, which is similar to the WT N.
leading to only partial conversion to cross-linked protein. europaea g values of 6.57, 5.09, and 1.97 and E/D of 0.03.10
Heme-Lysine Cross-Link Formation in cyt P460 Is Altogether, these data are consistent with a cross-link-
Intrinsic to the c′β Protein Fold. The cross-link-forming Lys containing protein species.
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Figure 11. UV/vis spectral time-course for the reaction of 8 μM Methylococcus capsulatus WT cyt c′β (A) or Phe61Lys cyt c′β (B) with 8
equivalents (64 μM) Li2O2. The red trace is before the addition of Li2O2, and the blue trace is the final spectrum after Li2O2 additions. Gray traces
represent scans every 0.2 min.

Figure 12. Summary of species observed during peroxide-driven cyt P460 maturation.

To extend our studies on cyt c′β, we expressed and purified

WT cyt c′β from Methylococcus capsulatus as well as its
■ CONCLUSIONS
We have demonstrated the necessity for peroxide in cyt P460
Phe61Lys variant. The WT protein did not react with peroxide.
maturation. This unique family of enzymes is primed for PTM
The purified Phe61Lys variant exhibited a UV/vis absorption
spectrum with a sharp Soret band at 407 nm and Q-bands at by virtue of the protein fold and the influence of this fold on
528 and 558 nm (Figure S21), in contrast to the broad Soret the structure of the bound c-heme. The assembled data
band of the WT cyt c′β and more similar to spectra of cross- strongly support that a peroxide-dependent PTM is involved in
link-deficient cyt P460 variants. Although the Lys variant was the maturation of cyt P460 proenzyme (Figure 12). A single
not purified as cross-linked protein like the Nitrosospira sp. intermediate accumulates during this maturation that has a
NpAv variant was, it did contain a small shoulder at 440 nm. Soret absorption maximum at 413 nm. This species can be
Upon reaction with peroxide, the peak at 440 nm grew in while generated by direct addition of exogenous peroxide to
the Soret band at 407 nm decayed (Figure 11). The product proenzyme and will decay to form catalytically competent
was a mixture of a 403 nm species and the 440 nm species. cross-linked cyt P460. Once peroxide binds, homolytic
This reactivity is reminiscent of the Arg44Ala mutant, which cleavage of the O−O bond leads to a high-valent heme
could form the cross-link but did not go to completion. Cyt c′β consistent with compound II and a hydroxyl radical, the latter
has minimal ruffling distortion of the heme comparable to the of which likely hydroxylates the γ-meso-carbon. We showed
Arg44Ala mutant, so it is possible that the ruffling deformation using an Arg44Ala mutation that ruffling is not required for
aids in cross-link formation but is not essential. However, the
cross-link formation, but that Arg44 plays an important role in
presence of a Lys in the cross-linking position is required for
directing reactivity to the γ-meso-carbon through a H-bond
reactivity with peroxide and subsequent cross-link formation in
these proteins. This suggests a role of Lys in either a hydrogen- network. Additionally, the installation of a Lys to the cross-
bond network or proton donation to the Fe-peroxo species to linking position in the related cytochrome c′β family, which has
aid in the homolytic cleavage of the O−O bond. The ability to the same fold as cyt P460 but does not contain the Lys cross-
install a heme-Lys cross-link in cyt c′β shows that the cross-link link, imbues reactivity with peroxide and leads to cross-link
formation is intrinsic to the fold and points to a further formation. This revealed that Lys does not simply wait in the
evolutionary relationship between the cyt c′β and cyt P460 active site to form the covalent attachment to the porphyrin
families. but also plays a role in O−O bond cleavage.
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■ ASSOCIATED CONTENT
* Supporting Information
sı
Contract No. DE-AC02-06CH11357. We thank Siddarth
Chandrasekharan for assistance with EPR data collection. We
The Supporting Information is available free of charge at also thank Robert W. Voland for assistance with rapid freeze-
https://pubs.acs.org/doi/10.1021/jacs.3c03608. quench experiments. The table of contents graphic was created
with Biorender.com.
SDS-PAGE, additional kinetics data, EPR spectra, 57Fe
Mössbauer spectra, EXAFS data and fit parameters,
heme planarity analysis, cyt P460 sequence logo (PDF) ■ REFERENCES
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K.M.L. gratefully acknowledges NIGMS for support in the
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