Pinpoint Chemical Modification of the Quinone-Access Channel of

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Pinpoint Chemical Modification of the Quinone-Access Channel of Mitochondrial Complex I via a Two-Step Conjugation Reaction Takahiro Masuya, Masatoshi Murai, Takeshi Ito, Shunsuke Aburaya, Wataru Aoki, and Hideto Miyoshi Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00612 • Publication Date (Web): 14 Jul 2017 Downloaded from http://pubs.acs.org on July 17, 2017

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Biochemistry

Pinpoint Chemical Modification of the Quinone-Access Channel of Mitochondrial Complex I via a Two-Step Conjugation Reaction

Takahiro Masuya, Masatoshi Murai, Ito Takeshi, Shunsuke Aburaya, Wataru Aoki, and Hideto Miyoshi*

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

*Corresponding Author: Hideto Miyoshi, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, E-mail: [email protected], Tel: +81-75-753-6119, Fax: +81-75-753-6408.

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ABBREVIATIONS BSA, bovine serum albumin; CBB, Coomassie brilliant blue R250; complex I, proton-translocating NADH-quinone oxidoreductase; DDM, n-dodecyl-β-D-maltoside; DOC, sodium deoxycholate; LDT chemistry, ligand-directed tosyl chemistry; MS, mass spectrometry; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SMP, submitochondrial particle.


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Biochemistry

ABSTRACT We previously showed that a bulky ring-strained cycloalkyne possessing a rhodamine fluorophore directly reacts (via strain-promoted click chemistry) with the azido group incorporated (via ligand-directed tosyl chemistry) into Asp160 in the 49 kDa subunit of complex I in bovine heart submitochondrial particles [Masuya et al. (2014) Biochemistry 53, 7816–7823].

This two-

step conjugation may be a promising technique for specific chemical modifications of the quinoneaccess channel in complex I by various molecular probes, which would lead to new methodologies for studying the enzyme.

However, since the reactivities of ring-strained cycloalkynes are

generally high, they also react with other nucleophilic amino acids in mitochondrial proteins, resulting in significant undesired side reactions.

To minimize side reactions and achieve precise

pinpoint chemical modification of 49 kDa Asp160, we investigated an optimal pair of chemical tags for the two-step conjugation reaction.

We found that instead of strain-promoted click

chemistry, Diels-Alder cycloaddition of a pair of cyclopropene incorporated into 49 kDa Asp160 (via ligand-directed tosyl chemistry) and externally added tetrazine is more efficient for the pinpoint modification.

An excess of quinone-site inhibitors did not interfere with Diels-Alder

cycloaddition between the cyclopropene and tetrazine.

These results along with the previous

findings (cited above) strongly suggest that in contrast to the predicted quinone-access channel modeled by X-ray crystallographic and single-particle cryo-electron microscopic studies, the channel is open or undergoes large structural re-arrangements to allow bulky ligands access close proximity with 49 kDa Asp160.


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INTRODUCTION NADH-quinone oxidoreductase (respiratory complex I) couples electron transfer from NADH to quinone with the translocation of protons across the membrane, which drives energyconsuming reactions such as ATP synthesis and substrate transport (1–3).

Complex I is the

largest enzyme of the mitochondrial respiratory chain and one of the major sources of superoxide production in mammalian mitochondria (4). The X-ray structures of entire complex I from Thermus

thermophilus

(5)

and

Yarrowia

lipolytica (6) were modeled at resolutions of 3.3 and 3.6 Å, respectively.

The structures of

mammalian complex I from bovine (Bos Taurus) (7) and ovine (Ovis aries) (8) hearts were recently determined

by

single-particle

cryo-electron

microscopy (Cryo-EM): the locations of all 45 subunits, including 31 supernumerary subunits, were assigned.

Despite these developments in

structural biology approaches, the mechanism underlying the coupling between electron transfer and proton translocation remains elusive. Ligand-directed

tosyl

(LDT)

chemistry,

which is based on the principle of affinity labeling, is a unique technique for chemical modifications of proteins (9, 10). acetogenin-driven recently

high-affinity

succeeded

in

By using

ligands,

specific

we

chemical

modifications of the quinone-access channel of intact complex I in bovine heart submitochondrial

Figure 1. Structures of AL derivatives (AL2, AL3, AL4, AL5, and AL6) and chemical tags (BODIPYtetrazine and TAMRA-DIBO) used in this study. The lengths of the BODIPY, tetrazine, TAMRA, and DIBO moieties, indicated by an arrow, mean the distance between two (C)-H atoms in their extended conformations. The lengths of the alkyl side chain are as follows: AL2, n = 3; AL3, n = 1; AL4, n = 1; AL5, n = 3; AL6, n = 3.


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Biochemistry

particles (SMPs) via LDT chemistry (11–13).

We demonstrated that Asp160 in the 49 kDa

subunit (49 kDa Asp160), which is located in the inner part of the channel (5–8), is specifically modified by different LDT chemistry reagents; for example, alkynylation [Asp160(COO)-(CH2)2CºCH] and azidation [Asp160(COO)-(CH2)3-N3].

This finding indicates that 49 kDa Asp160

elicits very strong nucleophilicity in the local protein environment (14).

(Note that 49 kDa

Asp160 corresponds to Nqo4 Asp139, 49 kDa Asp196, and NuoCD Asp325 in T. thermophilus, Y. lipolytica, and E. coli complex I, respectively). Ring-strained cycloalkynes directly react with an azido group (-N=N+=N-) via [3+2] azidealkyne cycloaddition (the so-called strain-promoted click chemistry) under physiological conditions (15, 16) (Figure S1).

Therefore, Asp160(COO)-(CH2)3-N3 may serve as a “handle” for

subsequent diverse chemical modifications by externally added ring-strained cycloalkynes (a second tag), which would lead to unique biochemical and/or biophysical studies on complex I.

In

160

order to characterize the reactivity of Asp (COO)-(CH2)3-N3 in intact complex I in SMPs, we examined whether this azido group directly reacts with an externally added ring-strained cycloalkyne possessing a rhodamine fluorophore (TAMRA-DIBO; approximately 13 × 20 Å, Figure 1), and found that a bulky TAMRA-DIBO directly react with Asp160(COO)-(CH2)3-N3 (12). Thus, this two-step conjugation may be a promising technique for specific chemical modifications of the quinone-access channel in complex I using various molecular probes.

The scheme of two-

step conjugation was illustrated in Figure S1 for clarity. Ring-strained cycloalkynes [e.g. dibenzocyclooctyne (DIBO)] may be useful molecular tags to be attached to Asp160(COO)-(CH2)3-N3; however, during the course of our previous study, it became clear that TAMRA-DIBO reacts not only with Asp160(COO)-(CH2)3-N3, but also with other nucleophilic amino acids in mitochondrial proteins because of its high reactivity; in particular, ADP/ATP carrier (12).

This undesired side reaction may be a common property of highly reactive

ring-strained cycloalkynes (17).

To develop unique chemistry-based techniques for the study of

complex I, improvements in the specificity of chemical modifications represent a challenging task (18).

To this end, an optimal pair of tags for each conjugation reaction (the first tag to be

conjugated to 49 kDa Asp160 and the second tag to be conjugated to the first tag) need to be screened out. In the present study, we synthesized various LDT ligands using acetogenin as a template, which possess different first tags to be attached to 49 kDa Asp160, and examined reactivities against 5 ACS Paragon Plus Environment

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externally added second tags.

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We found that a combination of the modification of 49 kDa Asp160

by cyclopropene [Asp160(COO)-(CH2)3-cyclopropene] via LDT chemistry and the subsequent conjugation of tetrazine via reverse-electron-demand Diels-Alder cycloaddition (19, 20) enables the precise pinpoint modification of this residue (“reverse-electron-demand Diels-Alder cycloaddition” is simply abbreviated to “Diels-Alder cycloaddition” throughout the text).

Using

this two-step conjugation, undesired side reactions were almost completely avoidable, even when using bovine SMPs as the experimental material.

On the basis of the results obtained in the

present study and previous findings (12), we discuss the structural features of the quinone-access channel in complex I.


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EXPERIMENTAL PROCEDURES Materials. Bullatacin and fenpyroximate were kindly provided by J. L. McLaughlin (Purdue University, West Lafayette, IN) and Nihon Nohyaku Co., Ltd. (Tokyo, Japan), respectively. Aminoquinazoline (AQ) and Δlac-acetogenin were the same samples as those used previously (21, 22).

Protein standards (Precision Plus Protein Standards and Precision Plus Protein Dual Xtra

Standards) for SDS-PAGE were purchased from Bio-Rad (Hercules, CA). was purchased from Jena Bioscience (Jena, Germany).

BODIPY-tetrazine

Other reagents were all of analytical grade.

Preparation of Bovine Heart Submitochondrial Particles (SMPs) SMP were prepared from isolated bovine heart mitochondria by the method of Matsuno-Yagi and Hatefi (23) and stored in buffer containing 250 mM sucrose and 10 mM Tris-HCl (pH 7.4) at -80 °C before being used.

The NADH oxidase activity in SMP was measured according to the

previously described procedures (24). The content of complex I in SMP was roughly estimated as the minimal amount of bullatacin required to completely inhibit the NADH oxidase activity because this inhibitor binds to the enzyme in a stoichiometric manner (25); the content of complex I in 1.0 mg of SMP proteins was estimated to be ~0.10 nmol. General Procedures for LDT Chemistry and Diels-Alder Cycloaddition LDT chemistry was conducted by incubating SMP (2.0 mg of protein/mL, 100−200 µL) with AL derivatives (AL3−AL6) in buffer containing 250 mM sucrose, 1 mM MgCl2, and 50 mM KPi (pH 7.4) at 37 °C for 24 h. Non-reacted AL6 residing in SMPs was removed, when necessary, by washing SMP with buffer containing 1% (w/v) bovine serum albumin (BSA) several times according to the previously described procedure (11). To conjugate BODIPY-tetrazine tag to the modified 49 kDa Asp160 via Diels-Alder cycloaddition, SMP samples treated with AL3−AL6 (above) were incubated with 0.1−30 µM BODIPY-tetrazine at 37 °C for 1.0 h.

The reaction was terminated by the addition of the 4×

sample buffers for SDS-, Clear Native (CN)-, or Blue Native (BN)-PAGE.

When the effects of

competitors (i.e. other quinone-site inhibitors) on Diels-Alder cycloaddition were investigated, cycloaddition was quenched by the addition of an excess of another cyclopropene derivative [(3(3-(methoxymethyl)cycloprop-1-en-1-yl)propyl)benzene shown in Figure S2, 100 molar-fold of 7 ACS Paragon Plus Environment

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BODIPY-tetrazine], to prevent the reaction between BODIPY-tetrazine and remaining modified 49 kDa Asp160 during the solubilization of mitochondrial proteins. Electrophoresis Proteins conjugated with BODIPY-tetrazine were resolved by Laemmli- or Schägger-type SDS-PAGE (26, 27).

CN-PAGE was conducted using a Native PAGE Novex Bis-Tris Gel

System with a 4−16% precast gel (Life Technologies, Carlsbad, CA) without Serva Blue G dye (CBB-G250).

To improve the resolution of oxidative phosphorylation enzyme complexes

(complexes I−V), 0.02% DDM and 0.05% DOC were added to the cathode buffer according to the conditions reported by Wittig et al. (28). BN-PAGE (11).

For the isolation of complex I, SMP were separated by

The migration pattern of fluorescent proteins was visualized using the model

FLA-5100 Bio-imaging analyzer (Fuji Film, Tokyo, Japan) with a 473 nm light source and LPB filter (510 nm) or 532 nm light source and LPG (575 nm) filter.

Data processing and the

quantification of fluorescence were conducted using Multi gauge software (Fuji Film, Tokyo, Japan). To analyze the BODIPY-attached 49 kDa subunit, it was partially purified by SDS-PAGE and electroelution (29).

The purified subunit was digested by lysylendopeptidase (Lys-C, Wako Pure

Chemicals, Osaka, Japan) or endoprotease Asp-N (Roche Life Science, Penzberg, Germany) in 50 mM Tris/HCl buffer (containing 0.1% SDS) or 50 mM NaPi buffer (containing 0.01% SDS), respectively (29).

The digests were separated on a Schägger-type SDS gel (16.5% T and 6% C

containing 6.0 M urea, ref. 27). Enrichment of the protein modified by AL6 SMPs (2.0 mg/mL, total 2.0 mg) were modified by AL6 (5.0 µM) at 37 ˚C for 24 h, and the modified proteins in SMPs were further conjugated with a cleavable biotin-SS-tetrazine (30 µM, see Figure S3) via Diels-Alder cycloaddition at 37 ˚C for 1 h.

The SMPs were solubilized with

1% (w/v) DDM, resolved by BN-PAGE, and complex I was isolated by electroelution using a model 422 Electro-Eluter (Bio-Rad). The recovered complex I was solubilized with 2% (w/v) SDS in a total volume of 100 µL, then diluted with Tris-buffered saline (1.0 mL) containing 1% (w/v) Triton X-100.

The biotinylated

protein (the 49 kDa subunit) in the isolated complex I was enriched using a streptavidin-agarose 8 ACS Paragon Plus Environment

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Biochemistry

CL-4B (Sigma-Aldrich, St. Louis, MO) according to the procedures described previously (11, 12). The specific capture/release procedures were monitored by SDS-PAGE (Figure S3), and the modified 49 kDa subunit was isolated as a single band on the SDS gel, which was subjected to “in gel” digestion with trypsin. The identification and characterization of the 49 kDa subunit were conducted using an LTQ Velos Orbitrap mass spectrometer equipped with Ultimate 3000 nano-LC (LC-MS, Thermo Scientific, Walthman) under the same experimental conditions as those reported previously (11). Data were analyzed using Proteome Discoverer 2.1 (Thermo Scientific) with Mascot 2.3 (Matrix Science, London, U.K.).

For the tryptic digestion, calbamidomethylation (Cys) was set as static

modification, and oxidation (Met) and the modification by AL6 [(C22H28N4O3S) at Asp, Glu and His (see Figure S4)] were set as dynamic modifications.


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RESULTS Synthesis of Acetogenin-Derived LDT Reagents (AL3–AL6) Strain-promoted click chemistry (azidocycloalkyne [3+2] cycloaddition) is efficient for chemoselective covalent conjugation of two chemical probes under physiological conditions (15, 16).

However, ring-strained

cycloalkynes, DIBO in our case, also react with

nucleophilic

amino

acids

in

mitochondrial proteins, as described in the Introduction.

Therefore, we herein selected

Diels-Alder cycloaddition between a ringstrained cycloalkene and tetrazine as an alternative conjugation reaction because of its very fast and selective reactivity (19, 20) and planned the experimental strategy as follows: a ring-strained cycloalkene (or cycloalkyne) is initially incorporated into 49 kDa Asp160 via LDT chemistry and tetrazine is sequentially added as the second tag to conjugate with the incorporated cycloalkene (Figures 2A and 2B).

We synthesized four acetogenin-

Figure 2. (A) The reaction mechanism of reverseelectron-demand Diels-Alder cycloaddition between tetrazine and cycloalkene. (B) Schematic representation of Diels-Alder cycloaddition between tetrazine tag and appropriate cycloalkenes or cycloalkyne.

derived LDT reagents (AL3–AL6) and BODIPY-tetrazine was used as the second tag (Figure 1).

The synthetic procedures of AL3–AL6

were described in Supporting Information. The inhibitory effects of AL3−AL6 and bullatacin were examined with the NADH oxidase activity in SMPs (30 µg of protein/mL).

The inhibitory potencies, in terms of IC50 values, of AL3,

AL4, AL5, AL6, and bullatacin were 2.7 (± 0.14), 5.9 (± 0.21), 3.5 (± 0.12), 2.1 (± 0.20), and 1.0 (± 0.15) nM, respectively.

These results indicate that all AL derivatives are slightly less active

than bullatacin, but still retain strong inhibitory effects at the single nM level.


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Biochemistry

Two-step Conjugation (LDT Chemistry and Diels-Alder Cycloaddition) with Complex I in SMPs SMPs (2.0 mg of protein/mL), which had been subjected to LDT chemistry using AL3, AL4, AL5, or AL6 (5 µM each) targeting 49 kDa Asp160, were incubated with 30 µM BODIPY-tetrazine at 37 °C for 1 h, followed by solubilization with 1% SDS for the SDSPAGE analysis. subsequent

If LDT chemistry and

Diels-Alder

cycloaddition

successfully occur, the conjugation product can be visualized as a fluorescent band on the gel.

As shown in Figure 3, a strong

fluorescent band at ~ 50 kDa (the 49 kDa subunit, as identified later) was determined only for the SMPs sample that had been treated with AL6. confirmed

that

conjugate

with

We preliminary

BODIPY-tetrazine the

cycloalkene

can or

cycloalkyne moiety of AL3−AL6 in water (containing 2% EtOH) to give cycloaddition

Figure 3. Modification of bovine heart SMP via a combination of LDT chemistry and Diels-Alder cycloaddition (two-step conjugation). Bovine heart SMPs (2.0 mg of protein/mL) were incubated with 5.0 µM of AL3, AL4, AL5, or AL6 at 37 °C for 24 h (LDT chemistry). SMPs were further incubated with fluorescent BODIPYtetrazine (30 µM) at 37 °C for 1 h to perform Diels-Alder cycloaddition. The proteins in SMPs were denatured in Laemmli’s sample buffer containing 2% SDS and 2.5% mercaptoethanol, separated on a 12.5% Laemmli type SDS gel, and subjected to CBB staining and fluorescent gel imaging. Data shown are representative of three independent experiments.

products, as illustrated in Figure 2B. Therefore, these results indicate that AL6 alone is efficient for the pinpoint modification of the 49 kDa subunit in combination with BODIPY-tetrazine.

We investigated which conjugation step,

LDT chemistry or Diels-Alder cycloaddition, is the cause of failed two-step conjugation for AL3, AL4, and AL5.

Since the solubilized 49 kDa subunit in SMP, which had been treated with AL3,

AL4, or AL5 (5 µM each) at 37 °C for 24 h, did not react with BODIPY-tetrazine, the first conjugation step (i.e. the modification of 49 kDa subunit via LDT chemistry) turned out to be unsuccessful for these derivatives.

The bulky tag moieties of AL4 and AL5 and the -NH-CO-

moiety of AL3 may be unfavorable for the attack by nucleophilic amino acid(s) in LDT chemistry.


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To examine the effects of other quinone-site inhibitors on the incorporation of a cyclopropene moiety via LDT chemistry, SMPs were incubated with AL6 (1.0 µM) in the presence of an excess amount (10 molar-fold) of bullatacin, fenpyroximate, rotenone, or Dlac-acetogenin, followed by conjugation with a fluorescent BODIPY-tetrazine cycloaddition.

via The

Diels-Alder incorporation

of

fluorescence into the 49 kDa subunit was completely suppressed by all the competitors tested

(Figure

4),

indicating

that

the

modification of the 49 kDa subunit by AL6 occurs inside the quinone-binding cavity.

Figure 4. Effects of quinone-site inhibitors on the incorporation of cyclopropene into the 49 kDa subunit. To examine the effects of quinone-site inhibitors on LDT chemistry, SMPs (2.0 mg of protein/mL) were incubated with AL6 (1.0 µM) in the presence of different complex I inhibitors, followed by conjugation with BODIPY-tetrazine (30 µM) via Diels-Alder cycloaddition. Proteins were separated on a 12.5% Laemmli type SDS gel and subjected to CBB staining and fluorescent gel imaging: a; negative control (without AL6), b; positive control (AL6 alone), c; AL6 plus bullatacin (10 µM), d; AL6 plus fenpyroximate (10 µM), e; AL6 plus rotenone (10 µM), f; AL6 plus Δlacacetogenin (10 µM). Data shown are representative of three independent experiments.


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Biochemistry

Identification of the Residue Modified by AL6 in the 49 kDa Subunit To identify the amino acid residue modified by AL6, we performed exhaustive digestion of the 49 kDa subunit, which was modified by AL6 (5.0 µM) and conjugated with BODIPYtetrazine, using Lys-C or Asp-N.

The digests

were resolved on a Schägger-type SDS gel to provide major fluorescent bands at ~8 kDa and ~12 kDa for Lys-C and Asp-N digestion, respectively (Figure 5).

Since these digestion

patterns were completely identical to those observed in the previous studies (11, 12, 14), in which Asp160 was specifically modified by different LDT reagents, the Lys-C and Asp-N digests were assigned to the regions Leu125– Lys176 (6.1 kDa) and Asp107–Gln201 (11.1 kDa), respectively.

These results strongly

suggest that endoprotease Asp-N failed to cleave the peptide bond between Leu159 and Asp160, resulting in a large digest with an apparent

Figure 5. Exhaustive digestion of the modified 49 kDa subunit. SMPs (2.0 mg/mL) were incubated with AL6 (5.0 µM) at 37 °C for 24 h, followed by conjugation with BODIPY-tetrazine (30 µM) via Diels-Alder cycloaddition at 37 °C for 1 h. Complex I was isolated by BN-PAGE on a 4–16% BN-PAGE gel and electroelution. The 49 kDa subunit was partially isolated by SDS-PAGE on a 12.5% Laemmli type SDS gel and electroelution, and digested with Lys-C or Asp-N, as described in the Experimental Procedures. The digests were analyzed on a Schägger type SDS gel (16.5% T and 6% C, containing 6.0 M urea), followed by fluorescent gel imaging. Data shown are representative of three independent experiments.

molecular mass of ~12 kDa (11, 12, 14). To pinpoint the amino acid residue modified by AL6, the SMPs treated with AL6 (5.0 µM) were conjugated with a clearable biotin-SS-tetrazine (Figure S3), and the modified complex I was isolated by a preparative BN-PAGE.

The biotinylated protein was captured and released by

immobilized streptavidin (Figure S3), followed by the digestion with trypsin.

The tryptic digests

were extensively characterized using LC-MS/MS by setting a specific chemical modification (C22H28N4O3S, exact mass of 428.1882, Figure S4) as a variable protein modification.

While the

enriched protein was identified as the 49 kDa subunit (33 peptides, 78.4% coverage, Figure S4), no modified peptides were detected in the region localized by the exhaustive digestion with Lys-C and Asp-N (Figure 5).

The candidate peptide Lys147-Arg174 (m/z 1103.21 or 1108.54,

corresponding to oxidation of one or two methionines, respectively) containing Asp160 was 13 ACS Paragon Plus Environment

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identified in an unmodified (free)-form.

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As discussed in previous photoaffinity studies including

our work (30, 31), the modified peptides are often determined in their free form, not in an adducted form, because of the cleavage of unstable modified sites under the analytical conditions.

This

instability may explain why we failed to determine the modified tryptic digest in the enriched 49 kDa subunit.

Thus, while we failed to pinpoint the amino acid residue modified by AL6 by mass

spectrometry, the modification must occur at 49 kDa Asp160, as observed for other acetogeninbased LDT reagents (11, 12, 14).


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Biochemistry

Optimization of Diels-Alder Cycloaddition between BODIPY-Tetrazine and Asp160(COO)-(CH2)3Cyclopropene SMPs (2.0 mg of protein/mL) were incubated with different concentrations of AL6 (0.1−5.0 µM), followed by conjugation with BODIPY-tetrazine (30 µM) via Diels-Alder cycloaddition. SMP samples were then solubilized with 1% (w/v) DDM and 1% (w/v) SDS for CN- and SDSPAGE analyses, respectively.

As shown in Figures 6A and 6B, cyclopropene was exclusively

incorporated into complex I among complexes I–V and the 49 kDa subunit among complex I subunits, respectively, in a concentration-dependent manner.

The saturation of fluorescence

increases in the bands was achieved at ~2.0 µM AL6 in both CN and SDS gels, which is estimated to be approximately 10 molar-fold of the complex I content. The previous results of the two-step modification of 49 kDa Asp160 in SMP via strainpromoted click chemistry using the pair of AL2 and ring strained cycloalkyne TAMRA-DIBO are shown in Figure 6C as a reference (12).

The fluorescent bands in the ~ 75 and ~ 30 kDa regions

are attributable to side reactions by TAMRA-DIBO.

A comparison of fluorescence images

between Figures 6B and 6C revealed that these side reactions were markedly reduced in the twostep modification via Diels-Alder cycloaddition.

We note that the protein corresponding to the

strong fluorescent band at the ~30 kDa region was identified as an ADP/ATP carrier by MALDITOF MS analysis of its tryptic digests; however, it was not possible to identify the proteins corresponding to ~75 kDa bands (12). To further reduce side reactions, we investigated the lowest possible concentrations of BODIPY-tetrazine for Diels-Alder cycloaddition.

Since BODIPY-tetrazine also reacts with

residual non-reacted AL6 in the SMPs suspension after LDT chemistry, higher concentrations of BODIPY-tetrazine are needed to conjugate with Asp160(COO)-(CH2)3-cyclopropene, resulting in an increase in the probability of side reactions.

Therefore, we attempted to remove non-reacted

AL6 using BSA. SMPs (2.0 mg of protein/mL) were subjected to LDT chemistry with 2 µM AL6 at 37 °C for 24 h, followed by washing with buffer containing 1% (w/v) BSA twice to remove nonreacted AL6.

SMP samples were then incubated with different concentrations of BODIPY-

tetrazine (0.1−30 µM) for 1 h.

Fluorescence intensity incorporated into Asp160(COO)-(CH2)3-

cyclopropene achieved saturation at ~3 µM BODIPY-tetrazine (Figure 7).

Compared to the

results shown in Figure 6B, in which 30 µM BODIPY-tetrazine was used, the side reaction with ADP/ATP carrier was significantly diminished under these experimental conditions. 15 ACS Paragon Plus Environment

The faint

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Page 16 of 27

fluorescent band at ~60 kDa was attributed to the reaction against residual BSA (marked by an asterisk).

Figure 6. Concentration dependency of the specific modification of 49 kDa Asp160 by AL6. SMPs (2.0 mg of protein/mL) were incubated with various concentrations of AL6 (0–5.0 µM) at 37 °C for 24 h, followed by conjugation with BODIPY-tetrazine (30 µM) via Diels-Alder cycloaddition. Treated SMPs were solubilized with 1% (w/v) DDM or 1% (w/v) SDS and resolved by a 4–16% CN gel (A) or 12.5% Laemmli type SDS gel (B), respectively, followed by CBB staining and fluorescent gel imaging. Data shown are representative of three independent experiments. As a reference, proteins in SMP, which were modified through a combination of AL2 and TAMRA-DIBO, were analyzed on a 12.5% Laemmli type SDS gel (C). Note that the gels in (C) were the same as those used in Figure 4 in ref. 12.


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Biochemistry

Figure 7. Effect of removing residual AL6 on the modification of 49 kDa Asp160 by AL6. SMPs (2.0 mg/mL) were incubated with AL6 (2.0 µM) at 37 °C for 24 h, and washed with a buffer containing 1% (w/v) BSA twice. SMPs samples were further incubated with various concentrations of BODIPY-tetrazine (0.1–30 µM) at 37°C for 1 h, and resolved by SDS PAGE on a 12.5% Laemmli type SDS gel, followed by fluorescent gel imaging and CBB staining. The protein marked with an asterisk is remaining BSA used for the removal of residual AL6. Data shown are representative of three independent experiments.


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Effects of Quinone-Site Inhibitors on the Conjugation between BODIPY-Tetrazine and Asp160(COO)-(CH2)3-Cyclopropene In the previous study (12), an excess of short-chain ubiquinone (Q2) as well as quinone-site inhibitors did not interfere with strain-promoted click chemistry between TAMRA–DIBO and Asp160(COO)-(CH2)3N3, except for bullatacin, a member of natural acetogenins. this

apparently

To examine whether unusual

effect

of

“competitor” is also seen with Diels-Alder cycloaddition between BODIPY-tetrazine and Asp160(COO)-(CH2)3-cyclopropene, the reaction was conducted in the presence of an excess

of

(quinazoline,

quinone-site

inhibitors Dlac-

fenpyroximate,

acetogenin, and bullatacin, 10 µM each). Quinazoline and fenpyroximate bind to the interface between the ND1 and 49 kDa subunits and between the 49 kDa and PSST subunits, respectively (24, 29). their

binding

locations

Although

are

slightly

Figure 8. Effects of quinone-site inhibitors on the conjugation between Asp160(COO)-(CH2)3-cyclopropene and BODIPY-tetrazine. SMPs (2.0 mg of protein/mL) were incubated with AL6 (2.0 µM) at 37 °C for 24 h, and washed twice with buffer containing 1% BSA to remove non-reacted AL6. SMPs samples were then incubated with 3.0 µM BODIPY-tetrazine at 37 °C for 30 min in the presence of bullatacin, aminoquinazoline, fenpyroximate, or Δlac-acetogenin (10 µM each). Diels-Alder cycloaddition was quenched by the addition of another cyclopropene derivative [(3-(3(methoxymethyl)cycloprop-1-en-1-yl)propyl)benzene, Figure S2] and subjected to Laemmli type SDS-PAGE using 12.5% gel (26), followed by CBB staining and fluorescent gel imaging. Data are representative of three independent experiments.

different, Dlac-acetogenin and bullatacin bind to the ND1 subunit, (32).

We preliminary

confirmed that 0.1 µM of each inhibitor completely blocks complex I activity under these experimental conditions.

Interestingly, quinazoline, fenpyroximate, and Dlac-acetogenin were

unable to interfere with the conjugation; however, bullatacin significantly, but not completely, suppressed the conjugation (Figure 8).

Bullatacin completely suppressed the conjugation

160

between TAMRA–DIBO and Asp (COO)-(CH2)3-N3 (12).

This more efficient suppressive

effect of bullatacin against TAMRA–DIBO may be due to the greater bulkiness of TAMRA–DIBO than BODIPY-tetrazine (Figure 1).


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Biochemistry

DISCUSSION A critical question regarding the mechanism of complex I is how the redox energy released by NADH-quinone oxidoreduction is transmitted to the proton pump modules located in the membrane domain.

While there is a consensus that the reduction of quinone at the predicted

quinone binding cavity plays a key role in this process, the mechanism responsible for the energy conversion remains elusive (1–3).

Therefore, studies are needed to elucidate the structural

features of the cavity as well as the mechanism underlying quinone reduction through various techniques (33, 34). We previously demonstrated that the bulky ring-strained cycloalkyne TAMRA-DIBO directly reacts with the azido group incorporated into 49 kDa Asp160 (12), which may be free from strict interactions (such as electrostatic interaction) arising from nearby residue(s) (13, 14).

Although

this finding suggested the possibility of diverse chemical modifications of the quinone binding cavity, the combination of the two conjugation reactions (LDT chemistry and strain-promoted click chemistry) was accompanied with significant undesired side reactions, as described in the Introduction.

The present study was conducted with a view to minimize side reactions and

achieve precise pinpoint chemical modification of 49 kDa Asp160.

We found that Diels-Alder

cycloaddition using the pair of cyclopropene incorporated into 49 kDa Asp160 [Asp160(COO)(CH2)3-cyclopropene] and an externally added tetrazine (BODIPY-tetrazine) is more efficient for pinpoint modification compared to strain-promoted click chemistry using the pair of an azido [Asp160(COO)-(CH2)3-N3] and ring-strained cycloalkyne (TAMRA-DIBO).

Side reactions were

almost completely avoidable in the case of Diels-Alder cycloaddition, except for a faint reaction with ADP/ATP carrier (Figures 6B vs. 6C).

The noticeable conjugation of BODIPY-tetrazine

with ADP/ATP carrier relative to other mitochondrial proteins may be due to the abundance of this protein in SMP rather than a specific interaction.

We note that the reaction of BODIPY-tetrazine

with ADP/ATP carrier may be minimized using the lowest possible concentrations of BODIPYtetrazine and SMPs sample, in which residual non-reacted AL6 was removed by BSA washing (Figures 6B vs. 7).

Moreover, the reactivities of tetrazine derivatives against the modified 49 kDa

Asp160 may generally be greater than those of ring-strained cyclooctyne derivatives because the former is less bulky than the latter (Figure 1).

Thus, the advantage of Diels-Alder cycloaddition

over ring-strained click chemistry may broaden the panel of second chemical tags.


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Page 20 of 27

On the other hand, investigations on the chemical reactivity of the first chemical tag incorporated into 49 kDa Asp160 will be valuable for characterizing the structural features of the quinone-access channel.

Viewed in this light, it is worth noting that excess amounts of quinones

(Q2 and Q6) and quinone-site inhibitors were unable to interfere with the conjugation reaction between Asp160(COO)-(CH2)3-N3 and bulky TAMRA-DIBO, except for bullatacin (a member of the acetogenin family) (12).

Although the types of modifications of 49 kDa Asp160 and

externally added second tags are both different, we herein obtained similar results for the reaction between Asp160(COO)-(CH2)3-cyclopropene and BODIPY-tetrazine (Figure 8).

Among the

inhibitors we tested, only bullatacin effectively interfered with the conjugation between the modified 49 kDa Asp160 and second tags, suggesting that the binding position of lipid-like acetogenins fairly differs from that of ordinary inhibitors (12, 32, 35). Upon considering the biochemical meaning of the results of the conjugation reactions described above, it should be reminded that Diels-Alder cycloaddition and strain-promoted click chemistry conducted in the present and previous studies (12), respectively, were performed against intact complex I in SMPs.

Also, TAMRA-DIBO and BODIPY-tetrazine have no specific binding

affinities to complex I; they randomly collide with the enzyme and only a fraction enters, by chance, into the cavity and reaches to the modified 49 kDa Asp160.

Taking these points into

consideration, it may be difficult to reconcile our results with the model of the quinone/inhibitoraccess channel derived from X-ray crystallography (5, 6) and Cryo-EM (7, 8), as discussed below. In bacterial complex I (5), the channel, extending from the membrane interior to the Fe-S cluster N2 (~30 Å long cavity), is a completely enclosed tunnel with only a narrow entry point (~8 Å diameter) for quinone/inhibitors.

The quinone-access channel in mammalian complex I modeled

by Cryo-EM was reported to be shorter and narrower than that in bacterial enzyme (7, 8).

If there

is only one entry point for quinone/inhibitors, as modeled, TAMRA-DIBO and BODIPY-tetrazine have to enter the narrow entry point and pass along the channel to react with the modified 49 kDa Asp160

[Asp160(COO)-(CH2)3-N3

and

Asp160(COO)-(CH2)3-cyclopropene,

respectively].

Although we considered this likelihood to be very low because TAMRA-DIBO and BODIPYtetrazine are much wider than the minimum diameter of the channel in the static structure (7), these bulky molecules did in fact conjugate with the modified 49 kDa Asp160.

Furthermore, if an

excess amount of quinone or inhibitor occupies the channel, these second tags may be unable to come into close proximity with the modified 49 kDa Asp160; however, this was not the case. 20 ACS Paragon Plus Environment

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Biochemistry

Quinazoline, fenpyroximate, and Dlac-acetogenin did not interfere with the conjugation between Asp160(COO)-(CH2)3-cyclopropene and BODIPY-tetrazine (Figure 8), though they completely blocked the access of AL6 to 49 kDa Asp160 (Figure 4).

Taken all together, it is strongly

suggested that the channel is open to allow bulky ligands access in close proximity to 49 kDa Asp160.

Our results also raise the question as to whether there is only one access path to this

residue.

In this context, Zhu et al. (7) suggested that there are alternative entrances in bovine

heart mitochondrial complex I, besides the entrance first identified in T. thermophilus, between the transmembrane helixes of the ND1 subunit, albeit narrower.

Since the planer ubiquinone ring (~6

Å across) is wider than the diameter of the putative channels (~2–3 Å), they also suggested that all channels in the static structure have to open to allow ubiquinone to enter. In ovine heart mitochondrial complex I determined by Cryo-EM (8), the inner part of the quinone-access channel around the critical amino acid residues 49 kDa His59 and Tyr108 is closed by a loop connecting two strands of the N-terminal b-sheet from the 49 kDa subunit, and this loop may prevent the quinone access close proximity to N2 cluster.

A similar model of the channel

was reported for the X-ray crystallographic structure of yeast mitochondrial complex I (6). These results have leaded the proposal that the models may represent the “deactive” state of the enzyme rather than a snapshot under catalytic turnover (3, 6, 8).

In the absence of respiratory substrates,

mitochondrial complex I exists in the “deactive” state and converts into the “active” state upon catalytic turnover in contrast to the always-active structure of the bacterial enzyme (36).

Since

complex I in bovine SMPs is in the deactive state under our experimental conditions because of the lack of respiratory substrates, our results suggest that the channel is open, even in the deactive state. We note that since the enzyme cannot be kept at an active state during the LDT chemistry (incubation at 37 °C for 24 h), we are unable to perform the chemical modifications during catalytic turnover.

On the other hand, we recently developed “decoupling” ubiquinones, the efficient

catalytic reduction of which in the quinone-access channel is insensitive to various quinone-site inhibitors and decoupled from proton translocation with bovine complex I (37, 38).

It is

conceivable that these ubiquinones accept electrons from an N2 cluster at slightly different position from that of typical short-chain ubiquinones such as ubiquinone-2.

Collectively, the

quinone/inhibitor-access channel in bovine complex I would undergo considerably large structural re-arrangements that enable the flexible accommodation of a wide range of ligands.


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Page 22 of 27

ACKOWLEDGEMENTS We thank Prof. Mitsuyoshi Ueda, Division of Applied Life Sciences, Kyoto University, for allowing us access to his LTQ Velos Orbitrap mass spectrometer as well as for his helpful advice on the experiments.

Funding This work was supported by a Grant-in-Aid for Scientific Research (Grant 26292060 to H. M. and Grant 15K07411 to M. M.) from the Japan Society for the Promotion of Science.

Notes The authors declare no competing financial interest.

SUPPORTING INFORMATION The syntheses of a key intermediate X1, AL3, AL4, AL5, AL6, (3-(3-(methoxymethyl)cycloprop1-en-1-yl)propyl)benzene, and biotin-SS-tetrazine. Figure S1: The two-step modification of the Asp160 in the 49 kDa subunit via a combination of LDT chemistry and strain-promoted click chemistry.

Figure S2: Structure of (3-(3-(methoxymethyl)cycloprop-1-en-1-yl)propyl)benzene.

Figure S3: Enrichment of the 49 kDa subunit modified by AL6. the 49 kDa subunit by LC-MS.


Figure S4: Characterization of

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Biochemistry

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Biochemistry

FOR TABLE OF CONTENTS USE ONLY

Pinpoint Chemical Modification of the Quinone-Access Channel of Mitochondrial Complex I via a Two-Step Conjugation Reaction Takahiro Masuya, Masatoshi Murai, Takeshi Ito, Shunsuke Aburaya, Wataru Aoki, and Hideto Miyoshi*


Pinpoint Chemical Modification of the Quinone-Access Channel of

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