Geometrical Changes in the Hemes of Bacterial Surface c-Type

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Biological and Environmental Phenomena at the Interface

Geometrical Changes in the Hemes of Bacterial Surface c-type Cytochromes Reveal Flexibility in their Binding Affinity with Minerals Yoshihide Tokunou, and Akihiro Okamoto Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b02977 • Publication Date (Web): 23 Oct 2018 Downloaded from http://pubs.acs.org on November 2, 2018

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Langmuir

Geometrical Changes in the Hemes of Bacterial Surface c-type Cytochromes Reveal Flexibility in their Binding Affinity with Minerals Yoshihide Tokunou1 and Akihiro Okamoto2* 1Department

of Applied Chemistry, The University of Tokyo, 7-3-1 Hongo, Bunkyo-

ku, Tokyo 113-8656, Japan

2International

Center for Materials Nanoarchitectonics, National Institute for

Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan

1

ACS Paragon Plus Environment

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ABSTRACT

2

Microbial extracellular electron transport occurs via the physical and electrical association

3

of outer-membrane c-type cytochromes (OM c-Cyts) with extracellular solid surfaces.

4

However, studies investigating the characteristics of cytochrome binding with solid

5

materials have been limited to the use of purified units of OM c-Cyts dissolved in solution,

6

rather than OM c-Cyts in intact cells, due to the lack of a methodology that specifically

7

allows for the monitoring of OM c-Cyts in whole-cells. Here, we utilized circular dichroism

8

(CD) spectroscopy to examine the molecular mechanisms and binding characteristics of

9

the interaction between MtrC, a unit of OM c-Cyts, in whole Shewanella oneidensis MR-

10

1 cells and hematite nanoparticles. The addition of hematite nanoparticles significantly

11

decreased the intensity of the Soret CD peaks, indicating geometrical changes in the

12

hemes in MtrC associated with their physical contact with hematite. The binding affinity

13

of MtrC estimated using CD spectra changed predominantly depending upon the redox

14

state of MtrC and the concentration of the hematite nanoparticles. In contrast, purified

15

MtrC demonstrated a constant binding affinity following a Langmuir isotherm, with a


2

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1

standard Gibbs free energy of −43 kJmol-1, suggesting that the flexibility in the binding

2

affinity of MtrC with hematite was specific in membrane-bound protein complex

3

conditions. Overall, these findings suggest that the binding affinity as well as the heme

4

geometry of OM c-Cyts are flexibly modulated in membrane complex associated with

5

microbe-mineral interactions.


3

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MAIN TEXT

2

1. INTRODUCTION

3

Dissimilatory metal-reducing bacteria have the ability to sense physical contact with

4

extracellular solid materials and thereby form an electrical connection. This connection

5

provides an external microbial respiratory chain that incorporates environmental minerals,

6

which enables microbes to generate energy and survive in a nutrient-limited environment

7

1.

8

driving the geochemical cycling of various elements, such as Fe, Mn, S, and C

9

variety of environmental settings

The electrons that are transported from the microbes reduce and dissolve minerals,

5-6.

2-4,

in a

This process can also be utilized with electrode

10

surfaces 7-8, which has signiﬁcant implications for the development of bioelectrochemical

11

technologies, such as microbial fuel cells 9-10 and electrode biosynthesis 11. The electrical

12

connections are made via cell-surface protein complexes known as outer membrane c-

13

type cytochromes (OM c-Cyts), which contain more than twenty heme redox centers

14

13.

15

solid materials have been extensively studied.

12-

Therefore, the molecular mechanisms underlying the interaction of OM c-Cyts and


4

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One of the best-studied proteins is MtrC, which is a deca-heme c-type cytochrome

2

that is a cell-exposed unit in OM c-Cyts within the metal-reducing bacterium Shewanella

3

oneidensis MR-1 (Figure 1) 14-15. Various crystallographic and biochemical studies have

4

revealed the molecular basis of the binding of MtrC and its homologues/paralogues (MtrF,

5

OmcA, and UndA) to minerals

6

minerals is proposed to be mediated by MtrC via its binding to minerals by way of near-

7

edge heme 15, 20. However, these studies have been limited to the examination of purified

8

protein dissolved in solution, instead of proteins within an intact cell. Given that MtrC in

9

an intact cell may interact with minerals differently than MtrC suspended in solution due

10

to the structural constraints imposed on the protein during the formation of OM c-Cyt

11

complexes embedded in a fluid phospholipid membrane, it is essential to monitor the

12

binding of MtrC protein to minerals within a living model system. Although the processes

13

involved in adhesion in intact S. oneidensis MR-1 have been studied using a variety of

14

techniques, such as atomic force microscopy 21-22, transmission electron microscopy 23-24

15

and attenuated total reflection infrared reflection spectroscopy

16

physical mechanisms underlying the interaction of the MtrC protein with minerals in intact

13, 16-19.

Rapid electron transport between microbes and

25-26,

the chemical or


5

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cells still remains unclear, mainly due to the complexity of the microbe-solid interface. S.

2

oneidensis MR-1 localizes a variety of other biomolecules to the cell membrane in

3

addition to OM c-Cyts. These biomolecules include lipopolysaccharides, extracellular

4

polymeric substances, and other membrane proteins

5

detection of the MtrC-mineral interaction in intact cells.

27,

which can interfere with the

6

Recently, we have developed whole-cell circular dichroism (CD) difference

7

spectroscopy, which can be used to measure the heme geometry of native MtrC proteins

8

in intact S. oneidensis MR-1 cells 28. The MtrC protein generates a Soret CD intensity that

9

is two orders of magnitude larger than that generated by mono-heme cytochrome, due to

10

the exciton coupling that takes place among the π-conjugated systems of the 10 hemes

11

in MtrC

12

oneidensis MR-1 strain lacking the gene for MtrC (ΔmtrC) from that obtained from a wild-

13

type (WT) strain allows for the measurement of the Soret CD signal generated solely by

14

the MtrC protein, with little interference from signals derived from other heme proteins 28.

15

Given that mechanical interaction with a solid surface may potentially induce

28.

Therefore, subtraction of the CD spectrum obtained from a mutant S.


6

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conformational changes in OM c-Cyts

2

information about the characteristics of MtrC-mineral binding. Here, we studied the

3

binding mechanism of MtrC in both intact cells and in the purified state using CD

4

spectroscopy. The CD spectra obtained in the presence and absence of minerals were

5

compared to obtain valuable molecular insights into the geometrical changes in the heme

6

centers of the MtrC protein that occur upon its interaction with minerals. Moreover, CD

7

spectra were obtained using various concentrations of mineral nanoparticles with both

8

oxidized and reduced MtrC to further quantify the binding affinity of MtrC with minerals.

9

We also discuss the insights gained during this study regarding the flexibility of heme

10

geometry and the binding affinity of MtrC associated with microbe-mineral interactions

11

and speculate on their implications for our understanding of the microbial ecophysiology

12

of the mineral surface and electron transport mechanisms.

13, 29-30,

changes in the CD signal may provide

13

14

2. EXPERIMENTAL SECTION

15

Sample preparation


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Shewanella oneidensis MR-1 bacteria were aerobically cultured in 15 mL of Luria-

2

Bertani [25 gL-1] medium with shaking at 160 rpm for 24 h at 303 K until the stationary

3

growth phase was reached. Subsequently, the cell suspension was centrifuged at 6,000

4

 g for 10 min, and the resultant cell pellet was washed twice with 15 mL of defined

5

medium (15 mM sodium succinate, 9.0 mM (NH4)2SO4, 5.7 mM K2HPO4, 3.3 mM KH2PO4,

6

2.0 mM NaHCO3, 1.0 mM MgSO4 7H2O, and 0.49 mM CaCl2; pH 7.4). Mutant strains that

7

lacked the mtrC, cymA, fccA, or PEC genes were constructed as previously described 31-

8

32.

9

by the manufacturer to be 20-40 nm, and this was confirmed by our own transmission

10

electron microscopic measurements. The MtrC protein was purified as previously

11

described

12

measurement of the UV-vis absorption spectrum (UV-2550, SHIMADU) of the air-oxidized

13

protein using an extinction coefficient of 1.26×106/M-1 cm-1 at 410 nm 33. The MtrC protein

14

solution was subjected to dialysis, and its pH was maintained at 7.5 with a 50 mM HEPES

15

buffer.

The diameter of the hematite nanoparticle (Skyspring Nanomaterials, Inc.) is reported

12, 14, 33,

and the concentration of the purified MtrC was determined via


8

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CD spectroscopy to monitor the inter-heme interactions in MtrC in the presence or

2

absence of hematite nanoparticles

3

CD spectroscopy was performed on a J-1500 (JASCO) CD spectrometer, and the

4

temperature was maintained at 293 K by an accessory Peltier element. The spectra were

5

collected using a Pyrex cuvette with a path length of 1.0 cm under conditions described

6

in a previous report (500 nm min-1 scan rate, 0.1 nm data pitch, and 5.0 nm bandwidth)

7

28.

8

optical density of 600 nm (OD600) of 1.33±0.02 was incubated for 40 min at 293 K after

9

the addition of hematite, as described in a previous report 21. The mixture containing the

10

cell suspension and hematite was stirred at 200 rpm in a Pyrex cuvette to prevent

11

precipitation during subsequent measurements. The CD spectra were integrated 9 times,

12

and the high tension (HT) voltage that was applied to the photomultiplier tube was

13

maintained below 550 V during every measurement to guarantee the validity of the data.

14

The CD spectra of the oxidized OM c-Cyts were obtained from a cell suspension under

15

aerobic conditions in the presence of 50 mM fumarate, while those of the reduced OM c-

To equilibrate the binding reaction, an S. oneidensis MR-1 cell suspension with an


9

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Cyts were obtained under anaerobic conditions in the presence of 30 mM lactate

28, 34.

2

The CD spectra of ΔmtrC strain bacteria were also obtained using the same conditions

3

to calculate the whole-cell differences in CD spectra for the wild-type (WT) and ΔmtrC

4

strains of S. oneidensis MR-1 28. The CD spectra of the purified MtrC protein at 0.20 μM

5

were obtained in a solution of 50 mM HEPES buffer with 100 mM NaCl at pH 7.4, using

6

conditions that were described in a previous report (200 nm min-1 scan rate, 0.5 nm data

7

pitch, and 1.0 nm bandwidth, with measurements taken 4 times) 28.

8

Scanning transmission electron microscopy (STEM) to visualize the association of S.

9

oneidensis MR-1 with hematite nanoparticles

10

Hematite nanoparticles at 13 mM were added into a cell suspension of S.

11

oneidensis MR-1 in defined medium with an OD600 of 1.33±0.02. After incubation for 40

12

min at room temperature, the microbe-nanoparticle suspension was collected by

13

centrifugation at 6000  g for 5 min and immediately fixed in a mixture of 4%

14

paraformaldehyde and 2.5% glutaraldehyde on ice. After fixation, we centrifuged the

15

sample at 6000  g for 5 min and washed with 50 mM HEPES ((2-[4-(2-hydroxyethyl)-1-


10

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piperazinyl] ethanesulfonic acid) buffer at pH 7.4 three times. The sample was

2

subsequently dehydrated in 25%, 50%, 75%, and 100% ethanol for 15 min each, then

3

directly applied onto a 200 mesh copper grid coated with carbon-sputtered ﬁlm (Nisshin

4

EM). The sample was dried overnight and imaged with a JEM-ARM200F microscope

5

operated at 80 kV.

6

Estimation of the free energy of binding of the reaction between purified MtrC protein and

7

hematite nanoparticles

8

We estimated the free energy of binding for the reaction between purified MtrC

9

and hematite nanoparticles using a sedimentation assay. The standard Gibbs free energy

10

(ΔG’) of the binding reaction was calculated based on the Langmuir adsorption model.

11

Hematite nanoparticles at 1.0 mM in 1.5 mL of solution containing 50 mM HEPES buffer

12

and 100 mM NaCl at pH 7.4 were mixed with various concentrations of MtrC protein and

13

then incubated at 293 K for 10 min to equilibrate the binding reaction. The mixture was

14

then centrifuged at 15,000  g for 3 min. The concentration of the unbound MtrC in

15

solution ([MtrC]/M) and the amount of MtrC bound to hematite (Γ/mol) was determined


11

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using UV-vis absorption spectroscopy. Within the framework of the Langmuir isotherm,

2

the standard Gibbs free energy, ΔG’, can be determined as follows:

1

1

3

[MtrC] Γ

4

ΔG’ = ―RT ∙ ln (𝑎) (Equation 2)

= ΓM ∙ [MtrC] + 𝑎 ∙ ΓM (Equation 1)

5

where ΓM is the theoretical maximum amount of MtrC that can be bound to hematite, and

6

a is the adsorption/desorption equilibrium constant. Since the sedimentation assay

7

provides the values of [MtrC] and Γ, the linear regression of [MtrC]/Γ against [MtrC] gives

8

ΔG’.

9

Estimation of the amount of MtrC-hematite complex at the intact cell surface in the

10

presence of various concentrations of hematite nanoparticles

11

When we assume that a single hematite nanoparticle binds with each MtrC protein

12

present on the intact cell surface, thereby altering the heme geometry of MtrC, the CD

13

peak intensity relates to the ratio of MtrC bound to the nanoparticles according to the

14

following equation:


12

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1

Langmuir

(1 ― θ) ∙ ∆𝜀0 +θ ∙ ∆𝜀1 = ∆𝜀𝑥 (Equation 3)

2

where θ is the ratio of MtrC bound to the hematite nanoparticle, and Δε0, Δε1, and Δεx are

3

the CD coefficients of unbound MtrC, bound MtrC, and a sample with a given

4

concentration of hematite, respectively. Thus, the ratio of MtrC bound to hematite

5

nanoparticles can be described by:

6

∆ε𝑥 ― ∆ε0

θ = ∆ε1 ― ∆ε0 (Equation 4)

7

In this study, Δε0 was set as 1.11×103/M-1 cm-1 (at 413 nm) and 1.97×103/M-1 cm-1 (at 421

8

nm) for MtrC in the oxidized and reduced states, respectively, as previously reported

9

Δε1 was estimated based on Figure 4 to be 0.60×103/M-1 cm-1 (at 413 nm) and

10

1.45×103/M-1 cm-1 (at 421 nm) for the oxidized and reduced states, respectively. To

11

quantify the coverage of MtrC during its association with a single hematite nanoparticle,

12

a hematite nanoparticle was redefined as one molecule, and the concentration was

13

recalculated as shown in Figure 5. Briefly, the number of hematite nanoparticles was

14

calculated based on the average volume of a nanoparticle (7.3×103/nm3) as estimated

15

using images obtained during transmission electron microscopy and subsequently

28.


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divided by Avogadro’s number to obtain an equivalent number of moles per liter

35.

The

2

total amount of MtrC protein at the intact cell surface was calculated at 413 nm using the

3

Soret CD intensity of the sample without hematite nanoparticles. The concentration of

4

unbound hematite nanoparticles was estimated using the difference between the

5

concentration of the added nanoparticles and that of MtrC bound to nanoparticles.

6

The curve derived from the Langmuir model using the identical ΔG’ of the binding

7

reaction between the purified MtrC proteins and the hematite nanoparticles that is

8

superposed on Figure 5 was calculated according to the following equation:

9

𝑎 ∙ [hematite]

θ = 1 + 𝑎 ∙ [hematite] (Equation 5)

10

where a is the constant calculated from Equation 1 that is related to the ΔG’ of the binding

11

reaction between the purified MtrC and the hematite nanoparticles obtained from

12

Equation 2, and [hematite] is the concentration of the hematite nanoparticles that were

13

not bound to MtrC.

14


14

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1

2

Langmuir

3. RESULTS AND DISCUSSION

Hematite induces geometrical changes in the heme centers in OM c-Cyts in intact cells

3

We first examined the inter-heme interaction in the MtrC protein in S. oneidensis

4

MR-1 cells in both the presence and absence of minerals using CD spectroscopy. In the

5

absence of minerals, we supplemented a cell suspension with an OD600 of 1.33±0.02 with

6

50 mM fumarate under aerobic conditions to oxidize the OM c-Cyts

7

contained two peaks around the Soret band at positions of 394 nm and 415 nm, as

8

reported previously 28 (Figure 2a). The peak at 415 nm is almost identical to that obtained

9

from the native MtrC protein (413 nm) 28, suggesting that this peak should be assigned to

10

the MtrC protein. However, the other peak, at 394 nm, was assigned to other units within

11

the OM c-Cyts, rather than the MtrC protein, via CD spectroscopy of the mutant strains.

12

The peak at 394 nm was completely absent in a mutant strain containing the complete

13

deletion of the periplasmic electron carrier, termed ΔPEC, which lacked the mtrA, mtrD,

14

dmsE, and SO4360 genes that encode the periplasm-facing units of the OM c-Cyts

15

(Figure 1 and S1). Deletion of the genes encoding representative intracellular cytochrome

34.

The CD spectra


15

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proteins (CymA or FccA: Figure 1), however, did not lead to the elimination of either of

2

the two Soret CD peaks (Figure S2), further suggesting that the Soret CD peaks observed

3

in Figure 2a originate from the OM c-Cyts.

4

We then added various concentrations of hematite nanoparticles with a diameter

5

of 20-40 nm into cell suspensions of S. oneidensis MR-1. Because the size of a

6

nanoparticle is slightly larger than that of the MtrC protein (approximately 9 nm×6 nm×4

7

nm) 14 and is two orders of magnitude smaller than the cell length of a S. oneidensis MR-1

8

bacterium, the hematite nanoparticles were potentially able to bind all of the MtrC proteins

9

on the cell surface. Both of the Soret peaks were clearly observed for all concentrations

10

that were tested, but the signal-to-noise ratio worsened as the concentration increased

11

(Figure 2). Although the addition of hematite nanoparticles at 2.0 to 4.8 mM had little

12

effect on the CD spectra, the Soret peak at 415 nm was greatly diminished upon the

13

addition of 9.1 mM hematite nanoparticles (Figure 2a). Since Soret peak intensity

14

corresponds to the degree of exciton coupling that occurs during inter-heme interactions

15

28, 36-38,

the change in the Soret CD profile strongly suggests that binding to hematite


16

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1

altered the heme geometry of MtrC in intact S. oneidensis MR-1 cells. In contrast, the

2

Soret CD peak at 415 nm for the S. oneidensis MR-1 cells showed little decrease upon

3

the addition of hematite after chemical fixation (Figure S3), strongly suggesting that the

4

significant decrease of CD signal intensity upon the addition of hematite before the

5

fixation resulted from substantial structure alteration in the MtrC protein. Specifically,

6

previous studies demonstrated that hematite nanoparticles with a diameter of 20-40 nm

7

uniformly bind to the surface of S. oneidensis MR-1 cells without aggregation

8

STEM images obtained in our study consistently showed that the nanoparticles were

9

evenly dispersed on the cell surface, while the dehydration and drying process prior to

10

measurement formed a few aggregations (Figure 3). Thus, conformational changes would

11

be expected to occur in the cell surface-exposed MtrC protein. The intensity of the Soret

12

peak at 394 nm also changed upon the addition of nanoparticles, suggesting that the

13

MtrC-hematite interaction induced geometrical changes in the heme moieties present in

14

PEC proteins as well. A Soret peak decrease was also observed in intact S. oneidensis

15

MR-1 cells containing reduced OM c-Cyts. We added 30 mM lactate to S. oneidensis MR-

16

1 cells that were subjected to anaerobic conditions, resulting in the shift of the Soret peaks

23-24.

The


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to longer wavelengths due to the reduction of the OM c-Cyts, as previously reported

2

(Figure 2b). The addition of 4.8 mM hematite led to a clear decrease in the intensity of

3

the Soret peak at 421 nm, which has an identical wavelength to that of the Soret CD peak

4

corresponding to the MtrC protein, supporting the conclusion that the presence of

5

hematite nanoparticles changes the heme geometry of MtrC in intact cells. Another peak,

6

at 411 nm, assigned to the PEC proteins (Figure S1) also fluctuated upon the addition of

7

hematite, further suggesting that physical contact between MtrC and the hematite

8

nanoparticles induces geometrical changes in the heme centers that comprise the OM c-

9

Cyts.

28

10

The use of whole-cell CD difference spectra to quantify geometrical changes in the heme

11

centers in MtrC

12

Since the data suggested that the changes in the whole-cell CD spectra originate

13

from the MtrC-hematite interaction, we quantified the intensity of the Soret peaks obtained

14

from the MtrC protein within intact cells. We previously reported that the difference

15

between the CD spectra obtained from WT and deletion mutant (ΔmtrC) strains of S.


18

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Langmuir

1

oneidensis MR-1 provides a CD profile of the native MtrC protein present in intact cells

2

28.

3

hematite nanoparticles. The Soret peak of the native, oxidized MtrC at 413 nm clearly

4

decreased in intensity upon the addition of 9.1 mM hematite, and the intensity increased

5

by almost 40% upon the addition of 23 mM hematite (Figure 4, S4 and S5), which

6

demonstrates that the binding of hematite to oxidized MtrC alters its heme geometry in

7

intact cells. Given that the local conformational changes in the amino acids surrounding

8

the heme centers cause significant changes in the shape of the Soret peaks, including

9

splitting effects

Thus, we obtained the difference in CD spectroscopy that occurred in the presence of

37, 39,

the decrease in CD intensity while the shape of the CD spectra for

10

MtrC in intact cells is maintained (Figure S5) supports the conclusion that the interaction

11

of bis-histidine residues with hemes is maintained during hematite binding. Consistently,

12

the HT voltage profiles recorded during CD measurements indicated an almost identical

13

Soret peak absorbance in both the presence and absence of hematite (Figure S6), which

14

suggests that the decrease in the intensity of the Soret CD spectra caused by the addition

15

of hematite stems from changes in the heme geometry, rather than from changes in the

16

interaction with bis-histidines or artefacts within the CD measurements such as down-


19

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Page 20 of 57

1

regulation of mtrC gene

2

aligned within the MtrC protein, and the heme on the edge, 5 or 10, is proposed to donate

3

electrons to minerals 15, 20. The contributions of heme 5 and 10 to Soret CD peak intensity

4

were estimated to be below 25% and 35% of the total signal intensity derived from the

5

MtrC protein, respectively

6

resulting from hematite binding. This finding indicates that the conformational changes

7

induced by hematite binding are not limited to the heme center that is adjacent to the

8

hematite nanoparticle but can take place in MtrC as a whole. Considering that the Soret

9

CD peak intensity is inversely proportional to the square of the distance between hemes

39.

Based on the crystal structure, the 10 heme centers are

28, 40,

which are less than that of the observed suppression

10

36, 41,

a decrease in the peak intensity suggests the occurrence of the unfolding of the

11

MtrC protein, leading to an increase in the distance between the heme centers. Notably,

12

the extent of the suppression of the Soret peak intensity originating from native, reduced

13

MtrC was approximately 25% in the presence of 23 mM hematite, which differs from that

14

of the oxidized species (Figure 4, S4 and S5). This suggests that the inter-heme

15

interactions within hematite-bound MtrC in a reduced state differ from those of oxidized

16

MtrC likely facilitating the electron transfer reaction. Overall, the whole-cell CD difference


20

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Langmuir

1

spectra indicate that contact between hematite nanoparticles and S. oneidensis MR-1

2

cells induces spatial arrangements of the deca-hemes in the MtrC protein.

3

We also examined the impact of hematite nanoparticles on heme geometry in the

4

purified MtrC protein. As we expected, the Soret CD peak obtained using 0.20 μM MtrC

5

dissipated upon the addition of hematite, suggesting that binding of the nanoparticles

6

causes geometrical changes in the heme centers of purified MtrC protein (gray plots in

7

Figure 4 and Figure S7). However, localization of proteins to the surface of nanovesicles

8

or nanoparticles flattens CD spectra 42, which distorts the quantification of changes in the

9

Soret CD spectra induced by the spatial arrangement of hemes. The extent of the

10

flattening effect depends upon the density of the proteins present on the surface of the

11

materials. Whole-cell CD spectra in the presence of a phospholipid/MtrC ratio of

12

approximately 2.0×103 (number of phospholipids per cell: ~2.0×107; number of MtrC

13

proteins per cell: ~1.0×104) show little flattening effect

14

MtrC protein is expected to densely cover the surface of a hematite nanoparticle, as

15

reported in OmcA

13;

42-45.

On the other hand, purified

in this case, it would be expected that the CD spectra are greatly


21

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Page 22 of 57

1

flattened. The decrease in Soret peak intensity derived from purified MtrC in the presence

2

of 7.0 mM hematite was approximately 90%; this exceeds the decrease that was

3

previously reported for an extreme example of the flattening effect, in which

4

bacteriorhodopsin that was densely embedded within a purple membrane at 75% (w/w)

5

content resulted in an 80% decrease in the CD signal intensity

6

decrease in the Soret CD peak intensity of MtrC was greater than that of the flattening

7

effect may suggest that the binding of hematite induces substantial arrangement of the

8

heme centers in both the purified MtrC protein and in the native MtrC protein present in

9

intact cells.

43.

The fact that the

10

The binding affinity of MtrC with hematite depends on the redox state of MtrC and the

11

concentration of hematite

12

Since the particle size of hematite is larger than that of the MtrC protein, it is

13

plausible that the number of hematite nanoparticles that can bind with a single MtrC

14

protein is limited to one. Based on the assumption that hematite binding leads to the

15

unfolding of MtrC, we plotted the ratio of hematite-bound MtrC and determined the


22

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Langmuir

1

concentration of hematite in solution (Figure 5), both of which were estimated using

2

whole-cell CD difference spectra. Since hematite nanoparticles are subject to little

3

aggregation (Figure 3) 23-24, a hematite nanoparticle was redefined as one molecule, and

4

the concentration was recalculated as described in the Experimental Section. For both

5

the oxidized and reduced native MtrC, the coverage of the hematite nanoparticle

6

increased as the particle concentration increased. In contrast, to cover 50% of the MtrC

7

protein in the oxidized and the reduced states, approximately 5.5×10-8/M and 2.5×10-8/M

8

hematite nanoparticles were required, respectively. The substantially lower concentration

9

of nanoparticles that was required for binding to reduced MtrC clearly indicates that the

10

affinity of MtrC for the nanoparticles is increased when this protein is in the reduced state.

11

Note that the difference between reduced and oxidized state is practically even larger,

12

because microbial iron reduction decreases the concentration of hematite nanoparticles

13

specifically in the presence of lactate. Given that complementary electrostatic interactions

14

are proposed to mediate binding between MtrF and hematite 19, redox-linked alteration of

15

the charge distribution in MtrC would be the main factor contributing to the change in the

16

binding affinity. Since the point of zero charge of hematite is approximately 8.5

27,

at pH


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Page 24 of 57

1

7.4, the positively-charged hematite would be more attracted to the reduced MtrC, which

2

would be more negatively-charged than the oxidized MtrC. Given that, unlike oxidized

3

MtrC, reduced MtrC requires contact with minerals to sustain microbial electrogenic

4

respiration, redox-linked enhancement of binding affinity is probably necessary for the

5

creation of an electrical connection between microbes and minerals, as suggested

6

previously 21-22.

7

In addition to the redox chemistry-based binding model, allosteric effects also likely

8

contribute to the curve profile shown in Figure 5. While the protein-mineral interaction is

9

generally described by the Langmuir adsorption model, which depicts a relationship

10

between coverage and mineral concentration that is characterized by a hyperbolic curve

11

13, 19,

12

Coverage of the native, oxidized MtrC protein by hematite was limited to only

13

approximately 5% in the presence of 3.0×10-8/M nanoparticles but was significantly

14

increased to approximately 70% in the presence of 6.0×10-8/M nanoparticles. This

15

dramatic increase in the bound ratio is typically observed for allosteric proteins, where the

the interactions of native MtrC were represented by curves with distinct shapes.


24

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Langmuir

1

binding of the first ligand enhances the affinity of a protein for the second ligand. To

2

examine whether this unique allosteric behavior is due to the characteristics of the MtrC

3

protein, the mechanisms of binding of the purified MtrC protein and hematite

4

nanoparticles were examined using a sedimentation assay (Figures 6 and S8). It was

5

found that the binding reaction proceeded according to a simple Langmuir adsorption

6

model with a standard Gibbs free energy (ΔG’) of −43 kJmol-1, demonstrating that the

7

presence of allostery is specific to the binding taking place in intact cells. In addition, the

8

superposed curve utilizing a ΔG’ value of −43 kJmol-1, shown in Figure 5, demonstrates

9

that a nanoparticle range on the same order of magnitude is present during binding,

10

suggesting that the chemical bond formed between MtrC and hematite is the same in

11

both the purified and native systems. Overall, it appears that the allosteric-like effect

12

observed upon binding of the native MtrC originates not in the characteristics of the MtrC

13

protein itself but in the specific functioning of MtrC when it is in membrane-bound protein

14

complex conditions.

15

The physiological implications of the flexibility of the binding affinity of MtrC


25

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1

Page 26 of 57

Given that the attachment of S. oneidensis MR-1 cells to mineral surfaces is

2

initiated by the formation of bonds between membrane proteins and hematite

3

flexibility of the binding affinity modulated by the redox state and the hematite coverage

4

of MtrC may assist in the attachment and detachment of cells from minerals. In particular,

5

the redox state of MtrC is closely related to the status of the intracellular respiratory chain;

6

thus, microbe-mineral interactions could be mediated by the metabolic condition of the

7

cells. When S. oneidensis MR-1 cells actively produce NADH from lactate present in their

8

environment, reduced MtrC tightly binds to the mineral surface, which allows for the

9

efficient regeneration of NAD+ by extracellular electron transport and subsequent 46-47.

26,

the

Furthermore, since OM c-Cyts are maintained in a

10

substrate-level ATP production

11

reduced state during electron transport 48-49, S. oneidensis MR-1 cells are able to adhere

12

stably onto mineral surfaces and continue substrate-level ATP production. Once the

13

electron donor, lactate, is starved, the metabolic reduction of OM c-Cyts stops; thus,

14

oxidized OM c-Cyts may assist in the detachment of S. oneidensis MR-1 cells from the

15

mineral surface to obtain lactate from elsewhere in their environment. This redox-linked

16

binding affinity allows microbes to reversibly and quickly switch between attachment to


26

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Langmuir

1

and detachment from the mineral surface, which would not be possible by utilizing the

2

much slower and intensive processes involved in the production and localization of

3

adhesive proteins and molecules; thus, this mechanism provides a method for microbes

4

to survive using electrogenic respiration.

5

For cells to achieve redox-dependent flexibility in their attachment and

6

detachment, electrostatically-controlled weak bond formation at the microbe-mineral

7

interface would be preferable to tight adhesion. Consistently, the ΔG’ of the binding

8

reaction between purified MtrC protein and hematite was estimated to be −43 kJmol-1

9

(Figure 6 and S7), which is much less than that of a typical bond formed during other

10

biomineralization interactions, such as that of amelogenin with hydroxyapatite (ΔG’ ~

11

−120 kJmol-1) 19, 50. Given that all of the reported ΔG’ values for the other components in

12

OM c-Cyts also suggest a weaker binding affinity than is typical for biomineral proteins

13

(MtrF: −55 kJmol-1; OmcA: −28 kJmol-1)13, 19, the observed features of MtrC may represent

14

characteristics that are common to mineral-microbe interactions. To verify the generality

15

of the redox-linked allosteric binding characteristics of microbe-mineral interactions, it


27

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Page 28 of 57

1

would be of interest to conduct whole-cell CD spectroscopy using other bacteria that

2

possess OM c-Cyts, such as Geobacter sulfurreducens, especially as a component of the

3

OM c-Cyts in Geobacter sulfurreducens, OmcS, exhibits a comparable Soret CD peak

4

intensity to that of MtrC

5

sulfurreducens PCA revealed large Soret CD peaks (Figure S9). Given that OmcS is a

6

major component of electrically conductive pili, which are required for direct electron

7

exchange between microorganisms 52-53, the use of this technique could shed light on the

8

structures involved in syntrophic respiration that is mediated by direct interspecies

9

electron transfer.

54.

Consistently, the whole-cell spectroscopy of Geobacter

10

However, the use of whole-cell CD spectroscopy with minerals has limitations that

11

depend on the size of the minerals. If the mineral size is comparable to or larger than that

12

of the microbe, only a proportion of the OM c-Cyts will bind to minerals, resulting in only

13

minute changes in the CD profile. Therefore, the application of whole-cell CD

14

spectroscopy would be limited to the study of microbe-nanoparticle associations, which

15

are not representative of most biomineral interactions that occur in nature. However, the


28

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Langmuir

1

data obtained from microbe-nanoparticle interactions may still aid in understanding the

2

interactions of microbes with large minerals. For example, when we consider the

3

interaction of S. oneidensis MR-1 cells with large minerals with rough surfaces, the area

4

of the surface with which an MtrC protein makes contact is expected to be small, which

5

is reflected by conditions involving a low nanoparticle concentration, as shown in Figure

6

5. As a result, the bond between the S. oneidensis MR-1 cell and the cell surface may be

7

lost due to the low binding affinity (Figure 5). The possibility of attachment and

8

detachment behavior being influenced by the nano- or microtopography of the mineral

9

surface is in good agreement with long-standing observations, such as that the roughness

10

of the mineral surface impedes the adhesion of S. oneidensis MR-1 cells 54 as well as the

11

dependence of the adhesiveness of cells to minerals on the crystal facets

12

implications may contribute to measures to control the formation of biofilms on solid

13

surfaces 56-58, which could also be used to accelerate the bio-reactions of metal-reducing

14

bacteria. Given that the attachment and detachment of microbial cells are controlled by

15

the surface property of minerals and electrodes

16

cellular components such as lipopolysaccharides and membrane proteins 25, 27, 54, it would

8

55.

Those

as well as the interactions of other


29

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Page 30 of 57

1

be of great interest to use whole-cell CD spectroscopy to further investigate the binding

2

characteristics of mutant strains that lack those components with various minerals.

3

The implications of the structural flexibility of MtrC for extracellular electron transfer

4

mechanisms

5

Our whole-cell CD spectroscopy experiments provided insights not only into the

6

factors affecting the binding affinity of OM c-Cyts but also into the flexibility of heme

7

geometry in OM c-Cyts. Although a homologous protein of MtrC, OmcA, has been

8

proposed to undergo structural changes induced by mineral binding

9

provided the first evidence of the spatial rearrangement of the hemes during the binding

10

of MtrC to hematite. Inter-heme interactions strikingly affect the kinetics of the electron

11

transfer reaction. Since hematite binding may cause the unfolding of MtrC in intact cells,

12

weakening the inter-heme interactions, the rate of electron transport is expected to

13

decrease; this is consistent with the relatively low electron transport rate that was

14

observed between OM c-Cyts and hematite compared with other minerals 59. This flexible

15

alteration of heme geometry suggests the potential for OM c-Cyts to regulate the kinetics

13, 29,

we have


30

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Langmuir

1

and pathways involved in electron transfer via the modulation of heme geometry based

2

on the components and/or facets present in the bound minerals. Indeed, resonance

3

Raman spectra obtained from purified MtrC have suggested that its binding with porous

4

indium tin-doped oxide electrodes induces alteration of the bis-histidine residues that are

5

adjacent to the heme centers, rather than a geometrical change in the hemes themselves

6

30.

7

supports the conclusion that MtrC forms different structures depending upon which

8

mineral it binds.

This distinct structural change in MtrC that was observed in our experiments further

9

Furthermore, the potential factors altering the heme geometry in OM c-Cyts are

10

not limited to the presence of minerals. Small angle x-ray scattering measurements of

11

purified OmcA proteins indicated that OmcA binding to a redox cofactor, flavin, induces

12

dimer formation and possible conformational changes

13

study using whole-cell CD difference spectra of S. oneidensis MR-1 cells indicated that

14

the inter-heme interactions in the MtrC protein are different in the purified system and the

15

native whole-cell system 28. To gain a true understanding of the mechanisms underlying

29.

Furthermore, another recent


31

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Page 32 of 57

1

the control of long-range electron transport over 100 Å in OM c-Cyts in living cells, it would

2

be essential to obtain molecular-level information about the formation of complexes

3

between OM c-Cyts and minerals, electrodes and redox cofactors.

4

5

4. CONCLUSION

6

In this work, we obtained the first evidence that microbe-mineral interactions

7

induce geometrical changes in the heme centers in the MtrC protein as well as in whole

8

OM c-Cyts, most likely associated with the unfolding of MtrC. These findings emphasize

9

the structural flexibility of MtrC, in which the kinetics and pathways of electron transport

10

potentially change depending on which molecules MtrC binds. To truly understand the

11

electron transport mechanisms in OM c-Cyts in living cells, it would be essential to obtain

12

molecular-level information about the formation of complexes between OM c-Cyts and

13

minerals, electrodes and redox cofactors. Furthermore, the binding affinity of MtrC with

14

hematite in intact cells was flexibly modulated by the redox state of MtrC and the ratio of

15

MtrC bound to the mineral. Since these parameters are linked to intracellular metabolism


32

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Langmuir

1

and extracellular environmental conditions, the flexible regulation of electrical

2

connections is expected to contribute to the ecophysiology of microbes that depend upon

3

interactions with minerals.

4

The flexibility of OM c-Cyts may provide further understanding of the mechanisms

5

underlying the electrical connections between microbes and minerals. Recent

6

experiments involving the use of deuterium with S. oneidensis MR-1 cells have suggested

7

that the binding of OM c-Cyts to flavins leads to the export of protons involved in the

8

electron transport reaction

9

show no clear hydrogen-bond networks that could be involved in conveying protons 14, 16.

10

Considering the structural flexibility of MtrC, it is plausible that a proton wire is formed as

11

a result of its binding to flavin. To elucidate the biochemical mechanisms underlying the

12

proton-coupled electron transport reaction occurring at the microbe-mineral interface,

13

which is most likely a common feature shared with other electrogenic microbes 62, it is of

14

great interest to obtain whole-cell CD spectra in the presence of cofactors such as flavins

15

and safranin

49.

52, 60-61;

however, the crystal structures of MtrC and OmcA

We hope that these insights contribute to the improved efficiency of


33

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Page 34 of 57

1

biotechnologies utilizing electrogenic respiration as well as to our fundamental

2

understanding of microbe-mineral interactions.

3

4

5

6

Figure 1. Location of OM c-Cyts, CymA, and FccA in S. oneidensis MR-1 cell. Heme-

7

containing proteins are illustrated in red color.

8


34

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Langmuir

1

2

Figure 2. Whole-cell CD spectra of S. oneidensis MR-1 cells subjected to aerobic

3

conditions in the presence of 50 mM fumarate (a) or 30 mM lactate under anaerobic

4

conditions (b). These spectra were obtained upon the addition of various concentrations

5

of hematite nanoparticles, as indicated by the legend. The arrows note the decrease of

6

the Soret CD peak at 415 nm (a) and at 421 nm (b) upon the addition of hematite

7

nanoparticles. The same tendency was reproduced in at least two separate experiments.

8


35

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Page 36 of 57

1

2

Figure 3. Bright-field STEM image of S. oneidensis MR-1 cells incubated under anaerobic

3

conditions for 40 min in the presence of 13 mM hematite nanoparticles and 30 mM lactate.

4

5

6

Figure 4. Soret peak intensity spectra obtained from whole-cell difference CD

7

representing native, oxidized MtrC at 413 nm (black plot) and reduced MtrC at 421 nm


36

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Langmuir

1

(red plot) as a function of hematite concentration. The gray plot represents the intensity

2

of purified, oxidized MtrC at 412 nm.

3

4

5

Figure 5. The fraction of native MtrC binding to hematite nanoparticles (θ) at various

6

concentrations of nanoparticles in solution. The black and red plots represent oxidized

7

and reduced MtrC, respectively. The gray line that is superposed on this graph represents

8

a hyperbolic curve using a ΔG’ value of −43 kJmol-1 following the Langmuir adsorption

9

model, which is identical to the binding affinity of purified MtrC protein that was estimated

10

in Figures 6 and S7. The θ was estimated from the Soret peak intensity of whole-cell CD

11

difference spectra, assuming that a single hematite nanoparticle binds with each MtrC


37

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Page 38 of 57

1

protein and alters the heme geometry. The concentration of hematite nanoparticles was

2

redefined to be one molecule. Further description of the calculations can be found in the

3

Experimental Section.

4

5

6

Figure 6. The amount of MtrC protein bound in the presence of 1.0 mM hematite

7

dispersed in 1.5 mL of solution (Γ/mol) at various concentrations of unbound MtrC protein

8

as estimated using a sedimentation assay. This provides a ΔG’ value of −43 kJmol-1 for

9

the binding of MtrC to the hematite surface. The data point in parentheses is not included

10

in the estimation of ΔG’.

11


38

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Langmuir

1

2

ASSOCIATED CONTENT

3

Supporting Information.

4

The Supporting Information is available free of charge.

5

Whole-cell circular dichroism (CD) spectra of mutant Shewanella oneidensis MR-1

6

strains, those of chemically fixed cells, those of Geobacter sulfurreducens PCA cells,

7

CD spectra of purified MtrC protein, HT voltage during CD measurement of WT, and

8

data analysis of sedimentation assay (PDF).

9 10

AUTHOR INFORMATION

11

Corresponding Author

12

*(Email: [email protected]). (A.O.).

13

Author Contributions


39

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1

Y.T. designed the study and conducted the experiments. Y.T. and A.O. prepared the

2

manuscript and have given approval to the final version of the manuscript.

3

Notes

4

The authors declare no competing financial interests.

Page 40 of 57

5 6

ACKNOWLEDGMENT

7

We thank Prof. Jeffrey A. Gralnick for kindly providing the ΔPEC and ΔfccA mutants and

8

Prof. Kenneth H. Nealson for ΔcymA mutant. We also express our thanks to Prof. Liang

9

Shi and Dr. Thomas A. Clarke for providing the purified MtrC protein, and to Prof.

10

Hiroyuki Noji for his advice. This work was financially supported by a Grant-in-Aid from

11

the Japan Society for Promotion of Science (JSPS) KAKENHI Grant Number 17H04969

12

to A.O. and 17J02602 to Y.T., the US Office of Naval Research Global (N62909-17-1-

13

2038) and the Japan Agency for Medical Research and Development

14

(17gm6010002h0002). We are grateful for the use of the scanning transmission

15

electron microscope at the National Institute for Materials Science Battery Research


40

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Langmuir

1

Platform in Japan. Y.T. is a JSPS Research Fellow and supported by JSPS through the

2

Program for Leading Graduate Schools (MERIT).

3 4 5 6

7

REFERENCES 1.

Myers, C. R.; Nealson, K. H., Bacterial Manganese Reduction and Growth with

Manganese Oxide as the Sole Electron-Acceptor. Science 1988, 240, 1319-1321.

2.

Nealson, K. H.; Saffarini, D., Iron and Manganese in Anaerobic Respiration -

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Environmental Significance, Physiology, and Regulation. Annu. Rev. Microbiol. 1994,

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48, 311-343.

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12

3.

Lovley, D. R.; Holmes, D. E.; Nevin, K. P., Dissimilatory Fe(III) and Mn(IV)

Reduction. Adv. Microb. Physiol. 2004, 49, 219-286.

4.

Risgaard-Petersen, N.; Revil, A.; Meister, P.; Nielsen, L. P., Sulfur, Iron-, and

13

Calcium Cycling Associated with Natural Electric Currents Running through Marine

14

Sediment. Geochim. Cosmochim. Acta 2012, 92, 1-13.


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1

5.

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Geometrical Changes in the Hemes of Bacterial Surface c-Type

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