1

Oxidase, Superoxide Dismutase, and Hydrogen Peroxide Reductase Activities of

Methanobactin from Type I and II Methanotrophs

Dong W. Choi1, Jeremy D. Semrau2, William E. Antholine3, Scott C. Hartsel4, Ryan C.

Anderson4, Jeffrey N. Carey2, Ashley M. Dreis4, Erik M. Kenseth4, Joel M. Renstrom4, Lori L.

Scardino4, Garrett S. Van Gorden4, Anna A. Volkert4, Aaron D. Wingad4, Paul J. Yanzer4,

Marcus T. McEllistrem4, Arlene M. de la Mora5, and Alan A. DiSpirito1*

1Department of Biochemistry, Biophysics and Molecular Biology and 5Psychology in Education Research

Laboratory, Iowa State University, Ames, IA, 50011-3211

2Department of Civil and Environmental Engineering, University of Michigan, Ann Arbor, MI, 48109-

2125

3Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI 53226

4Department of Chemistry, University of Wisconsin-Eau Claire, Eau Claire, WI 54702

Running title: Methanobactin Redox Properties

*CORRESPONDING AUTHOR FOOTNOTE: A.A. DiSpirito, Tel: 515-294-2944, Fax: 515-

294-0453

E-mail address: aland@iastate.edu

2

Abstract

Methanobactin (mb) is a copper binding chromopeptide that appears to be involved in

oxidation of methane by the membrane-associated or particulate methane monooxygenase

(pMMO). To examine this potential physiological role, the redox and catalytic properties of mb

from three different methanotrophs were examined in the absence and presence of O2. Metal

free mb from the type II methanotroph Methylosinus trichosporium OB3b, but not from the type

I methanotrophs Methylococcus capsulatus Bath or Methylomicrobium album BG8, were

reduced by a variety of reductants, including NADH and duroquinol, and catalyzed the reduction

of O2 to O2-.. Copper-containing mb (Cu-mb) from all three methanotrophs showed several

interesting properties, including reductase dependent oxidase activity, dismutation of O2-. to

H2O2 , and the reductant dependent reduction of H2O2 to H2O. The superoxide dismutase-like

and hydrogen peroxide reductase activities of Cu-mb were 4 and 1 order(s) of magnitude higher,

respectively, than the observed oxidase activity. The results demonstrate that Cu-mb from all

three methanotrophs are redox-active molecules and oxygen radical scavengers, with the

capacity to detoxify both superoxide and hydrogen peroxide without the formation of the

hydroxyl radicals associated with Fenton reactions. As previously observed with Cu-mb from

Ms. trichosporium OB3b, Cu-mb from both type I methanotrophs stimulated pMMO activity.

However, in contrast to previous studies using mb from Ms. trichosporium OB3b, pMMO

activity was not inhibited by mb from the two type I methanotrophs at low copper to mb ratios.

3

Key words: chalkophore; copper-binding compound; hydrogen peroxide reductase;

methanobactin; membrane-associated methane monooxygenase; methanotroph; Methylococcus

capsulatus Bath; Methylomicrobium album BG8; Methylosinus trichosporium OB3b; oxidase,

siderophore; oxygen radical scavenger, superoxide dismutase.

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1. Introduction

Methanobactin (mb) is a copper binding chromopeptide found in both the extracellular

and membrane fractions of many if not all aerobic methane oxidizing bacteria, or methanotrophs

[1-7]. The crystal structure of copper containing mb (Cu-mb) from Methylosinus trichosporium

OB3b showed the molecule represented a new class of metal binding compounds with a primary

sequence of N-2-isopropylester-(4-thiocarbonyl-5-hydroxy imidazolate)-Gly-Ser-Cys-Tyr-

pyrrolidine-(4-hydroxy-5-thiocarbonyl-imidazolate)-Ser-Cys-Met [6]. Copper coordination was

also unique with a dual S, and N coordination by 4-thiocarbonyl-5-hydroxy imidazolate (THI)

and 4-hydroxy-5-thiocarbonyl imidazolate (HTI) [6]. Recent studies have also suggested that

mb is a dynamic molecule in solution and appears to initially bind Cu(II) as a multimer, probably

a tetramer, via THI and Tyr [2, 3]. This initial binding is followed by a reduction to Cu(I) then

by coordination to HTI. Studies on the metal binding as well as on the solution and

thermodynamic properties of mb from Ms. trichosporium OB3b suggest the physiological

function of mb is that of a copper siderophore or chalkophore [2-9]

In addition to the extracellular fraction, Cu-mb is also found in the cell membrane

fraction [7]. In fact, Cu-mb was initially identified in association with the membrane-associated

or particulate methane monooxygenase (pMMO) and was originally proposed as a cofactor of

the hydroxylase component of the pMMO (pMMO-H) [1, 7, 10]. In this model, the pMMO was

a complex composed of three polypeptides (pmoA, pmoB, and pmoC), i.e. the hydroxylase

(pMMO-H) component plus 5 to 8 Cu-mb [1, 7, 10, 11]. Subsequent studies in other laboratories

have reported active preparations of pMMO-H with no evidence of Cu-mb [12-19]. The

reported activities of pMMO-H in the absence of Cu-mb, however, were low, only 2 - 25 % of

the reported activities for pMMO-H isolated with Cu-mb.

5

In addition to co-purification, the culture conditions used to stabilize cell free pMMO

activity also suggest an association between Cu-mb and pMMO [1, 10]. The high copper

conditions used to stabilize the pMMO results in increased concentrations of membrane-

associated Cu-mb. Cu-mb has also been shown to increase electron flow to the type II Cu(II)

centers of pMMO-H and to have superoxide dismutase activity suggesting secondary roles of

Cu-mb in methane catalysis by the pMMO [1, 10]. To examine the potential of mb in pMMO-H

stabilization or in electron flow to pMMO-H, the oxidase, superoxide dismutase, and hydrogen

peroxide reductase activities of mb was examined and compared to the effect of mb on pMMO-

H.

2. Experimental

2.1. Organisms, culture conditions and isolation of membrane fraction

Culture conditions for the isolation of mb from the spent media of Methylosinus

trichosporium OB3b were described previously [3]. Similar cultivation conditions were used for

the isolation of mb from Methylococcus capsulatus Bath, and from Methylomicrobium album

BG8. Methanobactin (mb) was prepared from the spent medium of Ms. trichosporium OB3b,

Mc. capsulatus Bath and Mm. album BG8 as previously described for Ms. trichosporium OB3b

[3, 10].

For the isolation of the washed membrane fraction and pMMO from Mc. capsulatus

Bath, cells were cultured in a batch reactor with a final CuSO4 concentration of 80 µM and

purified as previously described [10] with the omission of the second DEAE-Sepharose column

[20]. In addition to 2.4 Cu and 0.9 Fe atoms, 5 Cu-mb were associated with each αβγ subunit of

pMMO-H.

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2.2. Superoxide dismutase (SOD) activity assay

Superoxide anion radical (O2-⋅) was generated using phenazine methosulfate (PMS) in the

presence of a reductant (duroquinol or NADH). O2-⋅ production was determined via the reduction

of nitroblue tetrazolium (NBT) to blue formazan dye as described by Ramadan and El-Naggar

[21] with the following modifications. First the reactions were initiated by the addition NADH

(200 µM final concentration) or duroquinol to an aqueous solution of PMS (6 µM final

concentration), NBT (100 µM final concentration), and mb (10 µM final concentration). NADH

and duroquinol stock solutions were prepared as previously described [1]. Second, the copper to

mb ratio was varied between 0.01, 0.1, 0.25, 0.5, 0.75, 1.0, or 1.5 copper per mb and the reaction

mixture buffered with 10 mM 3-(N-morpholino) propane-sulfonic acid (MOPS) (final volume of

1 ml), pH 7.3. Copper controls were measured with equimolar CuSO4 to corresponding copper

to mb ratios. Rates of superoxide quenching were monitored by the rate of NBT reduction using

the molar extinction coefficient of 15 mM-1 cm-1 [22].

2.3. Anaerobic nitroblue tetrazolium (NBT) reduction

Direct reduction of NBT by mb was monitored under anaerobic conditions using NADH

as a reductant. Reagents were prepared in a Coy anaerobic chamber (95% Argon, 5% Hydrogen)

and checked for oxygen contamination with anaerobic indicator strips (Oxoid Ltd., Hampshire,

UK) prior to use. Duplicate anaerobic reaction mixtures containing 100 µM NBT and 10 µM mb

were prepared in anaerobic cuvettes. Reactions were initiated by addition of an anoxic NADH

solution with a gas-tight syringe. After 10 min incubation at room temperature, one of the

duplicates was purged with atmospheric air to introduce O2.

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2.4. Oxidase and hydrogen peroxide reductase (HPR) activities.

Reductant dependent oxidase activity was monitored by oxygen consumption rates at

room temperature (approximately 21°C). Reactions were initiated by adding NADH (0.5mM

final concentration) or duroquinol (0.5mM final concentration) to reaction mixtures containing

either 50 or 100 µM mb. Oxygen concentrations were monitored with either a YSI model 5300

biological oxygen monitor (Yellow Springs Instruments Co., Yellow Springs, OH), a ISO2

oxygen monitor equipped with a 2 mm diameter OXELP electrode (World Precision Instruments

Inc., Sarasota, FL), and/or by the fluorescence based oxygen sensing system (Ocean Optics Inc.,

Dunedin, FL). Frequent comparisons were made between each monitoring system to ensure the

results from each oxygen monitoring were similar, if not identical. The presence of hydrogen

peroxide in the reaction mixture was determined by the addition of catalase (20 – 400 nM final

concentration).

Hydrogen peroxide reductase (HPR) activity was also determined by the reduction of

catalase activity in reaction mixtures containing 0.5 mM NADH, 10 nM catalase, and 50 µM mb

at 21°C. The reaction mixture in the 1 ml oxygen-sensing chamber was purged with argon until

stable anaerobic base readings were achieved, then H2O2 (2 mM final concentration) was injected

to initiate the reaction. Oxygen evolution rate from the catalase reaction was measured and the

reduction of oxygen evolution rate in the presence of mb was considered as HPR activity. Data

were analyzed using linear fitting in Igor Pro 5.03 (Wavematrics Inc., Lake Oswego, OR).

pH changes associated with HPR activities by Cu(II) and Cu-mb were monitored in

separate reaction mixtures. In reaction mixtures examining HPR activity by Cu(II), duplicate

reaction mixtures containing 58 mg·ml-1 bovine serum albumin were used to approximate the

buffering capacity of mb.

8

2.5. Analysis of oxygen uptake rates.

The oxygen consumption data were analyzed to determine the reaction kinetics. A power

law kinetic model was selected for this analysis, i.e., for a constant-volume batch reactor:

!kC

dt

dC"= (1)

where C is the oxygen concentration, t is time, k is the rate constant, and α is the reaction order

[23]. To obtain an equation for oxygen concentration as a function of time, Eq. (1) was

integrated, giving

( )[ ]( )!! !""

""=111

01 ktCC (2)

where C0 is the initial oxygen concentration. To find the values of k and α, Microsoft Excel’s

solver tool was used to minimize the sum of the squared residuals between the measured oxygen

concentration, from the raw data, and the predicted oxygen concentration, from Eq. (2). That is,

the quantity

( )[ ]( )[ ]!=

"" """N

i

imiktCC

1

2111

01

## # (3)

was minimized by varying k and α.

Prior to analysis, data were smoothed by averaging instrument readings over 30-second

intervals to reduce error in data regression due to noise.

2.6. Methane oxidation activity, protein determination, electron paramagnetic resonance

(EPR) spectroscopy, UV-visible absorption spectroscopy, and metal analysis.

9

Methane oxidation activity, protein, EPR spectroscopy, copper and iron determinations

were carried out as described previously [1, 3, 10, 24]. The copper binding capacity of mb from

Ms. trichosporium OB3b was examined by incubation of mb in the presence of a molar excess of

CuCl2 for 5 min followed by isolation using Sep-Pac cartridges as previously described [1].

2.7. Molecular-mass determinations.

Molecular masses of mb samples from Ms. trichosporium OB3b, Mc. capsulatus Bath

and Mm. album BG8 were determined on an Agilent Technologies Model 6210 Time-of-Flight

Liquid Chromatograph/Mass Spectrometer (TOF LC/MS) using electrospray ionization at the

interface between LC and MS. Exact masses (± 5 ppm) were determined for mb as a negative

ion using four co-ionized calibration compounds.

2.8. X-ray photoelectron spectroscopy (XPS).

XPS was preformed on a model Phoibos-150 hemispherical analyzer (SPECS Scientific

Instruments, Sarasota, FL) as previously described [25], using an Al Ka X-ray source operated at

150W. To estimate X-ray-induced reduction of Cu(II) in Cu/mb complexes, samples were dried

onto graphite substrates, introduced into the instrument and aligned by monitoring the N XPS.

Total X-ray exposure time was monitored, and the Cu 2p3/2 was measured by scanning from 920

to 950 BE; 20 or 30 scans were signal-averaged. Spectra were fit to two peaks (using CasaXPS

software), and the area of Cu(I) and Cu(II) determined as a function of X-ray exposure time.

The Cu(II) areas were normalized against the total XPS area for Cu and scaled to the

experimental Cu/mb ratio (based on solution stoichiometry).

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3. Result and discussion

3.1 Extracellular concentration of mb.

The highest yields of mb in the spent media of all three methanotrophs were obtained

from nitrate minimal salts media (NMS) amended with 0.2 µM CuSO4, but the yields varied with

Ms. trichosporium OB3b showing the highest concentrations (35 – 60 mg·l-1) followed by Mc.

capsulatus Bath (18 - 24 mg·l-1) and Mm. album BG8 (1 – 10 mg·l-1).

3.2. Superoxide dismutase-like activity

Cu-mb has been shown to have superoxide dismutase-like (SOD) activity [10]. To

determine if the stimulatory and/or inhibitory effects of mb on pMMO activity were related to

SOD-like activity, such activity by mb was determined at different Cu to mb ratios (Fig. 1). The

results showed a positive correlation between these two activities (Fig. 1 and Table 1). In fact,

the potential production of superoxide with mb from Ms. trichosporium OB3b was observed at

the Cu to mb ratios found to be inhibitory to pMMO activity (see 3.8. Effect of mb on pMMO-H

activity in the washed membrane fraction and 3.9. Effect of mb on purified pMMO-H

preparations). The unexpected increased dye reduction rates suggested mb either reduced O2 to

O2-. or reduced nitroblue tetrazolium (NBT).

3.3. Oxidase activity by mb

To determine if mb reduced O2 to O2-., oxygen uptake rates were monitored at different

Cu to mb ratios (Figs. 2a and 3). The mb from all three mb samples showed Cu-dependent

oxidase activity with either NADH or duroquinol as a reductant. The mb from Ms. trichosporium

OB3b also showed Cu-independent oxidase activity. When NADH was used as a reductant with

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mb from Ms. trichosporium OB3b, highest oxidase activities were observed at high (c.a. > 0.75

Cu per mb) and low Cu (c.a. < 0.2 Cu per mb) to mb ratios, and lowest activity at Cu to mb ratio

of 0.5 Cu per mb (Fig. 3A). However, when duroquinol was used as a reductant, an inverse

response to Cu was observed with the highest oxidase activity observed at 0.5 Cu per mb (Fig.

3B).

Catalase was added to oxidase reaction mixtures under steady state conditions to

determine if hydrogen peroxide was an end product of the oxidase reactions by mb (Figs. 2A, 3C

and 3D). At low Cu to mb ratios (i.e. < 0.2 Cu per mb), the addition of catalase to the reaction

mixture resulted in the release of 0.5 O2 for every O2 consumed (Figs. 2A, 3C, 3D and 4). The

ratio of O2 consumed versus O2 released following catalase addition under low Cu to mb ratios

was similar if not identical to the predicted values for a one electron reduction of O2 (reaction 2)

followed by the chemical or biological dismutation of superoxide to hydrogen peroxide (reaction

3).

1. XH2 + mb → X2+ + mb-2

2. mb-2 + 2O2 → 2O2-. + mb

3. 2O2-. + 2H+ → H2O2 + O2

4. Catalase Reaction: H2O2 → H2O + 0.5O2

At higher Cu to mb ratios, the O2 released following catalase addition was less than the

predicted 0.5 O2 produced per O2 consumed (Figs. 3 and 4). As described below, reductant

dependent hydrogen peroxide reductase (HPR) activity of mb accounted for the difference

between the predicted and observed O2 evolution following catalase addition.

3.4. Oxygen uptake rate

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In longer-duration oxygen uptake experiments the reaction order α was observed to be

2/3, and approximate rate constants for the oxygen uptake reactions were calculated by assuming

that the reaction order was 2/3 regardless of Cu to mb ratio. For Cu to mb ratios of 0.0, 0.1, 0.25,

0.5, 0.75, and 1.0, the approximate 2/3-order reaction rate constants k were calculated to be 0.067

± 0.005, 0.074 ± 0.006, 0.001 ± 0.005, 0.005 ± 0.003, 0.006 ± 0.002, and 0.038 ± 0.003

µM1/3•min-1, respectively.

3.5. Reduction of nitroblue tetrazolium by mb

Although capable of generating superoxide, the rates of oxidase activity were too slow to

account for the increased rates of NBT reduction observed with mb from Ms. trichosporium

OB3b at low Cu to mb ratios (Figs. 1 and 2A) suggesting direct NBT reduction by mb. To assay

for direct reduction of NBT by Cu-mb, the reaction was monitored under anaerobic conditions

(Fig. 2B). Consistent with direct dye reduction, reactions initiated under anaerobic conditions

were identical to those monitored under aerobic conditions and the rates comparable to the

increased rates of dye reduction observed in the SOD experiments (Figs. 1 and 2B).

Unexpectedly, NBT reduction under anaerobic conditions stopped in anaerobic samples pulsed

with dioxygen. Oxygen consumption was observed in pulsed samples suggesting a change of

electron acceptor from dye to dioxygen.

3.6. Hydrogen peroxide reductase activity.

Hydrogen peroxide reductase activity by mb or Cu-mb was monitored by the quenching

of the rate of oxygen evolution by catalase (Figs. 4 and 5). As observed with oxidase activity,

the mb from both type I methanotrophs followed the general trend of copper dependent HPR

13

activity, while the mb from Ms. trichosporium OB3b showed highest HPR activities at copper

concentrations less than or greater than 0.5 Cu per mb (Fig. 5). To determine if the HPR

activities of Cu-mb differed from free copper, the oxygen and pH was monitored in the presence

and absence of a reductant. In the absence of a reductant, the addition of H2O2 to a CuCl2

solution resulted in the production of one proton and 0.5 dioxygen per H2O2 oxidized (Fig. 6),

which is consistent with reactions 5 – 10.

5. Cu2+ + H2O2 → Cu+ + O2H + H+

6. Cu2+ + O2H → Cu+ + O2 + H+

7. Cu+ + H2O2 → Cu2+ + OH + OH-

8. Cu+ + OH + H+ → Cu2+ + H2O

9. OH- + H+ → H2O

10) Sum: 2H2O2 → O2 + 2H2O + 2H+

Oxygen evolution stopped following the addition of a reductant to free copper (Fig. 6A),

with an associated increase in pH (Fig. 6B), which would be consistent with the reaction

sequence outlined in reactions 11 – 15.

11. 2Cu2+ + NADH + H+→ 2Cu+ + NAD+ + 2H+

12. Cu+ + H2O2 → Cu2+ + OH + OH-

13. Cu+ + OH + H+ → Cu2+ + H2O

14. OH- + H+ → H2O

15. Sum: H2O2 + NADH + H+ → 2H2O + NAD+

In contrast to free copper, in the absence of a reductant, no oxygen evolution was

observed in reaction mixtures containing with Cu-mb and H2O2 (Fig. 6A). In the presence of a

reductant, H2O2 was reduced by Cu-mb (Fig. 6A) with no pH changes (Fig. 6B). The results

14

suggest that H2O2 reduction by Cu-mb proceeds in the absence of reactive oxygen species

associated with free copper. As a control, bovine serum albumin, a known copper binding

protein, was added to copper reaction mixtures at equal (wt/wt) concentrations to that of mb

used in the Cu-mb reaction mixture. The bovine serum albumin and copper containing reaction

mixtures showed similar if not identical pH changes to the free copper reaction mixtures

suggesting the absence of a pH change in Cu-mb resulted from a different reaction mechanism,

not to the buffering capacity of mb.

3.7. Redox properties in the absence of an external reductant.

Previously, we reported XPS results demonstrating that mb will reduce Cu(II) to Cu(I)

upon binding the metal ion [3]. Initial efforts to quantify the number of Cu(II) ions reduced by

mb lead to inconsistent results. This was thought to possibly arise from the reduction of Cu(II)

to Cu(I) by secondary electrons generated in the sample from the X-ray source as previously

reported for transition metals. A cursory examination by monitoring the sample over time

indicated minimal such reduction, but follow-up studies showed that a loss of intensity in the

Cu(II) line with the corresponding increase in Cu(I) does occur (results not shown). However,

further changes that could be attributed to the reduction of Cu(I) to Cu(0) were never observed

regardless of X-ray exposure. This X-ray-induced reduction created a problem in measuring the

amount of mb-induced reduction of copper. Experiments were therefore undertaken to factor out

the instrument-induced systematic error by measuring copper spectra over the course of one to

two hours. If the number of secondary electrons excited by the X-ray source are assumed to be

constant, the decreasing peak area for Cu(II) followed pseudo-first-order kinetics. In order to

measure the loss of Cu(II), the Cu 2p3/2 spectra were fit to two peaks (using the program

15

CasaXPS) and the area of Cu(I) and Cu(II) were determined as a function of X-ray exposure

time. Normalizing the measured Cu(II) areas to the total area for all Cu oxidation states and

scaling areas to the Cu per mb ratio (based on stoichiometry) permitted the Cu(II) signal decay

to be measured. A fit of the data to a single exponential using Igor Pro allowed the

determination of Cu(II) in the absence of X-ray irradiation at t=0. Following correction, the

results showed that the mb from Ms. trichosporium OB3b will reduce approximately 2 Cu2+ to

2Cu+, but only when exposed to Cu2+ concentrations > 2 Cu2+ per mb. In general the corrected

results are similar to previous studies [3, 6], and suggest that Cu2+ will displace Cu+ bound to mb

or the presence of multiple Cu2+ binding sites.

In contrast to copper coordination determined in the crystal structure [6], the EPR spectra

of mb from Ms. trichosporium OB3b suggest Cu(II) coordination of 2N2O or 3N1O, with no

detectable sulfur coordination (Fig. 7). The quality of the signals was quite good as indicated by

the resolution of a 65Cu shoulder from the 63Cu signal. The improved quality of mb samples was

the result of improvements in the purification procedure [10]. The results suggest a change in

copper coordination from 2N2S or 3N1S to 2N2O during oxygen catalysis and may represent the

fraction of the population involved in oxygen turnover. The signal at g = 1.98 could be either

from iron or a copper dimer or both (Fig.7). Iron concentrations in the samples examined were

0.02 Fe per mb and is consistent with the intensity of the iron-EPR signal. However, the EPR

spectra of ferric saturated mb samples only showed a narrowing of the g = 4.3 peak suggesting

coordination [2], but not the g = 1.98 signal observed in these samples.

In an attempt to generate a 2N2S signal, the EPR spectra of Cu-mb were examined under

anaerobic conditions. At Cu to mb ratios of 0.5 only a small decrease in the intensity of the Cu

and potential Fe signals were observed following incubation under anaerobic conditions for 30

16

min (Fig. 7, trace A). The absence of oxygen in EPR samples was verified with resazurin

indicator strips. At higher copper to mb ratios, no EPR active copper or iron was observed

suggesting metal reduction (Fig. 7, trace B). Taken together the results indicate that electron

flow within mb differs at different Cu to mb molar ratios.

3.8. Effect of mb on pMMO-H activity in the washed membrane fraction

The effects of mb from the type I methanotrophs Mc. capsulatus Bath and Mm. album

BG8 and from the type II methanotroph Ms. trichosporium OB3b on pMMO activity in the

washed membrane fraction of Mc. capsulatus Bath varied with the reductant, with the source of

mb, well as with the Cu to mb molar ratio (Fig. 8). When NADH was used as a reductant, mb

from all three methanotrophs stimulated pMMO activity regardless of the final copper to mb

ratio (Fig. 8A; Table 1). Mb is a copper-chelator or chalkophore [3, 6] so the final copper to mb

following addition of mb to the washed membrane fraction is difficult to determine. However,

with the exception of mb from Mc. capsulatus Bath, stimulation was maximal at or near the

predicted ratio for the dimer or monomer copper coordination state [3].

When duroquinol was used as the reductant, the effect of mb on pMMO activity in the

washed membrane fraction from Mc. capsulatus Bath was more complex (Fig. 8B). As

previously observed [10], mb from Ms trichosporium OB3b stimulated pMMO activity at Cu to

mb ratios ≥1 Cu per mb but was inhibitory at lower Cu to mb ratios (Table 1). In contrast, little

to no inhibition at low Cu to mb ratios was observed with the mb from the two type I

methanotrophs with optimal stimulation of pMMO activity between 0.5 and 1 Cu per mb.

3.9. Effect of mb on purified pMMO-H preparations.

17

Duroquinol has been shown to be the preferred reductant in purified pMMO samples

from most laboratories [1, 7, 12, 14, 15, 19, 26, 27] (Table 2). However, in contrast to previous

studies from this laboratory, the addition of Cu-mb failed to stimulate pMMO activity using

duroquinol as a reductant. The higher initial activity in pMMO preparations used here may

explain the lack of stimulation. The pMMO samples examined here contained approximately 6

Cu-mb per αβγ subunit of pMMO-H. Also in contrast to previous preparations from this

laboratory, NADH could serve as an electron donor to the pMMO without co-purification of

detectable NADH-dehydrogenase [1]. The rate of propylene oxidation by pMMO samples with

NADH was only 50% of the activity observed with duroquinol (Table 2). However the activity

using NADH as a reductant could be increased to approximately 75% of the activity observed

with duroquinol by the addition of 2 molar equivalents of Cu-mb per pMMO-H monomer

suggesting the involvement of Cu-mb in electron flow to pMMO-H. Assuming electron flow

from Cu-mb to pMMO-H, the propylene oxidation rates are consistent with the oxidase activities

of mb (Tables 1 and 2).

As in previous studies [1, 10, 28], the type II Cu center of pMMO-H was approximately

50% reduced when isolated under anaerobic conditions (Fig 9, trace A, and data from 77K, not

shown). The addition of dioxygen, duroquinol, and the substrate propylene results in nearly

complete reduction of both the iron [20] and type-II copper [1, 12, 26, 28, 29] centers suggesting

the rate of reduction of pMMO-H was higher than the rate of propylene oxidation (Fig. 9, trace B

and Fig. 10). The addition of dioxygen, NADH, and propylene also results a similar reduction of

the type II Cu signal (Fig. 9, trace C). However, new signals, possibly a low spin Fe(III) signal

with a low field g-value of 2.48 and a diiron signal with g-values of about 2.07, 1.98, and 1.89

were observed (Fig. 9D). The low spin Fe(III) signal is tentatively assigned to the diiron site

18

where only one Fe(III) occupies the site. Although only 20% of the diiron signal was associated

with g-values of 2.07, 1.98, and 1.89, this signal was assigned to the mixed valence Fe(II)Fe(III)

state [20]. The results suggest the mixed valence Fe(II)Fe(III) center was re-oxidized before the

type II Cu(II) center (Fig. 10). The results also suggest electron flow using NADH as a reductant

in cell free systems is:

NADH → Cu-mb → Cu-pMMO-H → Fe-Fe-pMMO-H

Where Cu-pMMO-H is the type II Cu(II) center of the pMMO and Fe-Fe-pMMO is the diiron

center of pMMO-H [20].

3.10. Summary and concluding remarks

In addition to SOD-like activity reported previously [1, 11], the results presented here

shows Cu-mb also exhibits reductant dependent-oxidase and –hydrogen peroxide reductase

activities. Numerous studies have shown the accidental formation of O2-. when dioxygen

adventitiously withdraws an electron from redox components of respiratory chains [30-35].

Studies have also shown that many redox active biomolecules will reduce NBT in the absence of

dioxygen [36-39]. Both activities are consistent with the results presented here and in previous

studies [1, 7, 10] showing the Cu-mb can enhance electron flow to the pMMO. The inhibitory

effects of mb from Ms. trichosporium OB3b on pMMO activity at low Cu to mb ratios is

probably due to the accumulation of reactive oxygen species. Similar inhibitory effects have

been observed with respiratory vesicles of Escherichia coli when incubated with a reductant and

atmospheric dioxygen [40-44].

With respect to stimulation of the pMMO, previous studies have shown full reduction of

the type II Cu(II) center of the pMMO in the washed membrane fraction was only observed

19

under anaerobic conditions following the addition of both a reductant, NADH or duroquinol, and

Cu-mb, suggesting Cu-mb is involved in electron flow to the pMMO [10]. The results presented

here using NADH as a reductant also suggest Cu-mb is involved in electron flow to the pMMO.

As observed with in purified pMMO samples, reduction rates for Cu-mb with duroquinol were

higher than with NADH [1, 10, 12, 27]. In addition, assuming Cu-mb can serve as an electron

donor to the type II Cu(II) site in pMMO-H, the spectral differences observed when NADH is

used in place of duroquinol as an electron donor would be consistent with a high re-oxidation

rate for the diiron center of the pMMO [20]. Figure 10 summarizes our model for Cu-mb

mediated electron from the respiratory chain to the pMMO and electron flow within this enzyme.

In contrast to Cu(I) coordination shown in the crystal structure [6], the EPR spectra of mb

from Ms. trichosporium OB3b suggests 2N2O or 3N1O coordination rather than 2N2S. This

result suggests Cu bound by mb may change coordination in the presence of dioxygen and/or

with the oxidation state of copper.

20

4. Abbreviations

Cu-mb methanobactin copper complex

HPR hydrogen peroxide reductase

HTI 4-hydroxy-5-thiobarbonyl imidazolate

mb methanobactin

MMO methane monooxygenase

NBT nitroblue tetrazolium

P Pearson correlation

pMMO membrane-associated or particulate methane monooxygenase

pMMO-H hydroxylase component of the membrane-associated methane

monooxygenase

PMS phenazine methosulfate

r significance (two-tailed)

sMMO soluble methane monooxygenase

SOD superoxide dismutase

THI 4-thiocarbonyl-5-hydroxy imidazolate

XPS X-ray photoelectric spectroscopy

21

Acknowledgements

This work was supported by the Department of Energy grants 96ER20237 to ADS and

WA and DE-FC26-05NT42431 to JDS and by a grant from the Battelle BioScience Alliance of

Iowa to ADS.

22

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26

Figure Legends

Fig. 1. Superoxide dismutase (SOD) activity of Cu () and of mb from Mc. capsulatus Bath

(), Ms. trichosporium OB3b () and Mm. album BG8 () at different Cu to mb ratios.

Fig. 2. A. Oxygen trace as measured with a 2mm diameter OXELP oxygen senor following the

addition of NADH and either of mb () or Cu-mb () from Ms. trichosporium OB3b,

followed by the addition of 20, 100, 200 or 400 nM catalase. B. Reduction of nitroblue

tetrazolium by Cu-mb from Ms. trichosporium OB3b using NADH as a reductant under

aerobic () and anaerobic conditions (). Arrow indicates the addition of air to

anaerobic reaction mixture.

Fig. 3. Oxidase activity of Cu () and of mb from Mc. capsulatus Bath (), Ms. trichosporium

OB3b () and Mm. album BG8 () at different Cu to mb ratios using (A) NADH or

(B) duroquinol as a reductant. Oxygen evolution following the addition of catalase to

reaction mixtures containing mb from Mc. capsulatus Bath (), Ms. trichosporium

OB3b () and Mm. album BG8 () at different Cu to mb ratios using (C) NADH or

(D) duroquinol as a reductant.

Fig 4. Proposed reaction mechanism for oxidase and superoxide dismutase (SOD) activities of

mb. Cu-mb, and Cu in the presence of NADH and for the competition between catalase

and hydrogen peroxide reductase (HPR) for H2O2.

Fig. 5. Hydrogen peroxide reductase activity of Cu () and of mb from Mc. capsulatus Bath

(), Ms. trichosporium OB3b () or Mm. album BG8 () at different Cu to mb ratios

using NADH as a reductant.

27

Fig. 6. A. Oxygen trace of anaerobic solutions of Cu(II) or Cu-mb following the addition of

H2O2 (1 min), followed by the addition of 400 nM catalase. B. Change in proton

concentration in anaerobic solutions of Cu(II) ( and ), or Cu-mb from either Mc.

capsulatus Bath (), Ms. trichosporium OB3b () or Mm. album BG8 () following

the addition of H2O2 () and with the addition of H2O2, and NADH (,, , ).

Fig. 7. A. X-band EPR spectra at 77K of 5 mM mb containing 0.5 Cu per mb (a) and 0.8 Cu per

mb (b) under aerobic and anaerobic conditions. Experimental conditions: modulation

frequency, 100 KHz; modulation amplitude, 5G, time constant 100 ms, microwave

frequency 9.219 GHz, and microwave power 5 mW.

Fig. 8. Effect of mb from Mc. capsulatus Bath (), Ms. trichosporium OB3b () and Mm.

album BG8 () at different Cu to mb ratios on propylene oxidation by the washed

membrane fraction of Mc. capsulatus Bath using (A) NADH or (B) duroquinol as a

reductant. Dotted line marks the pMMO activity of in the washed membrane fraction

before the addition of mb.

Fig. 9. X-band EPR spectra at 7.6 K of 640 µM pMMO sample as isolated under anaerobic

conditions (A), following the addition of 2 mM duroquinol, 2 ml air and 2 ml propylene

(B), following the addition of 2 mM NADH, 2 ml air, and 2 ml propylene (C). (D) trace

C minus trace B. Experimental conditions: modulation frequency, 100 KHz; modulation

amplitude, 5G, time constant 100 ms, microwave frequency 9.632 GHz, and microwave

power 50 mW.

Fig. 10. Working model for Cu-mb mediated electron flow from the respiratory chain to the

metal centers of the pMMO from M. capsulatus Bath. Size of Cu-mb in relation to

28

pMMO was increased for illustration purposes. Abbreviations, X, periplasmic electron

donors; Q, ubiquine-8.

Table 1

Pearson correlations of pMMO activity in the washed membrane fraction from Mc. capsulatus Bath grown at 80 µM CuSO4 amended

media to the oxidase, H2O2, reductase and superoxide dismutase activities of mb Mc capsulatus Bath (Mc), Ms trichosporium OB3b

(Mt), and Mm. album BG8 (Ma) at different Cu to mb ratios.

NADH Duroquinol

pMMO Oxidase H2O2 Reductase Oxidase H2O2 Reductase Superoxide Dismutase

Mc Mc Mt Ma Mc Mt Ma Mc Mt Ma Mc Mt Ma Mc Mt Ma

NADH

Mc -0.74 -0.64 -0.81 0.21 -0.49

Mt -0.55 0.73 -0.77 0.73 0.83

Ma 0.97 0.92 0.92 0.99 0.99

Duroquinol

Mc 0.53 0.67 0.45 0.91 0.93

Mt -0.68 0.79 -0.71 0.98 . 0.93

Ma 0.90 0.84 0.85 0.93 0.95

Numbers in bold: correlations are significant at the 0.05 level (2-tailed).

Numbers in bold in shaded box: correlations are significant at the 0.01 level (2-tailed).

Table 2.

Base reaction rates of superoxide dismutase, oxidase and H2O2 activities of mb and of

pMMO activity as isolated and in the washed membrane fraction from Mc. capsulatus

Bath grown at 80µM CuSO4 amended media.

Propylene Oxidation

Reductant SOD Oxidase HPR Membrane pMMO

(O2.-· min-1·mb-1) ( O2·min-1·mb-1) (H2O2· min-1· mb-1) (propylene oxide⋅min-1·mg protein-1)

- 3000 - - - -

NADH - 0.1 5 240 70

Duroquinol - 0.7 ND 70 140