JSPS-NSF International Collaborations in Chemistry (ICC Program)

-Chemistry of Life Processes-

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JSPS-NSF International Collaborations in Chemistry (ICC) Project

Key words (where possible, include links to learn more):
Biological iron-sulfur cluster (ancient modular metallo-cofactor)LinkIconLinkIcon
Rieske [2Fe-2S] protein and cytochrome bc1/b6f complexLinkIconLinkIconLinkIconLinkIconLinkIcon
Electron spin resonance (EPR) spectroscopyLinkIconLinkIcon
High-resolution pulsed EPR spectroscopyLinkIconLinkIconLinkIcon
Protein crystallizationLinkIcon and X-ray crystallographyLinkIconLinkIcon
Single-crystal EPRLinkIconLinkIcon
Low-temperature resonance Raman spectroscopyLinkIconLinkIcon
Site-directed mutagenesisLinkIconLinkIconLinkIcon
Uniform and site-specific stable isotope (15N, 13C, 57Fe) labelingLinkIconLinkIconLinkIconLinkIcon
Heterologous overproduction of thermophile metalloenzymes

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Outcome of the ICC Project Research

-for general readers-

* A New Approach to Describe the Active Site Features in Biological Metalloenzymes

Understanding how the protein frame moiety controls the chemical properties of the biological active-site metal center in a protein macromolecule could not only provide fundamental scientific knowledge, but can also help to develop new protein-based catalysts with predictable functions.

Modern magnetic resonance-based spectroscopy (two-dimensional pulsed electron spin resonance) allows for direct probing of the hydrogen bonding interactions around the active-site metal center with the protein frame in a purified metallo-protein (produced with selective amino acid isotope labeling using a new Escherichia coli auxotroph expression host strain(s)), even when only a moderate or low resolution three-dimensional structure of the protein of interest is available.

Project Outcome

We have developed a novel experimental approach, combining biochemical and magnetic resonance-based spectroscopic techniques, to probe (i) the hydrogen bonding interaction network around an active-site metal center in a biological metalloprotein macromolecule, even when a high-resolution three-dimensional structure is not available, and (ii) the electronic structure and long-distance, outer-sphere chemical bond interactions around an active-site metal center in a biological metalloprotein macromolecule using selective cysteine isotope labeling technique.

Impact and Benefits

A new experimental approach is directed at describing the structural details of an active-site environment in a metallo-protein macromolecule containing a stable amino acid isotope (such as 15N and 13C) label(s) introduced around the (paramagnetic) active site. The chemical reactivity of a biological active-site metal center bound to the protein moiety is controlled by numerous factors including the hydrogen bonding network with the protein frame. Viewing this network typically requires a high-quality three-dimensional (3D) structure of the target protein at a high resolution where the bond lengths and angles can be defined with reasonable accuracy. However, 3D structures of many proteins of biological importance are available at moderate or lower resolutions where the hydrogen bond interactions can only be assessed ambiguously. The developed approach, combining biochemical and (two-dimensional pulsed electron spin resonance) spectroscopic techniques, can define the specific hydrogen bonding interactions with the protein frame even in these cases. A set of new “amino acid auxotroph” expression host strains of the lab-use Escherichia coli has been prepared, which can be used for stable amino acid isotope labeling of a variety of proteins in a cost-effective manner, for basic studies as well as industrial applications.

The chemical reactivity of a biological active-site metal center is also controlled by long-range outer-sphere interactions with the protein frame. The extent of outer-sphere electron spin transfer in a metalloprotein macromolecule was analyzed by residue specific assignments of the cysteine 13C(beta) couplings near the biological [2Fe-2S] cluster cofactor provide insight for the first time into the relative convalencies of the iron-cysteine bond interactions that play an important role in the overall redox potential and electron transfer reactivities of the biological iron-sulfur cluster cofactors. It is likely that Nature takes advantage of the inherent asymmetric spin density distribution resulting from the local fine-structure around the bioloical (reduced) [2Fe-2S] cluster to facilitate better electronic coupling preceding electron transfer, which also reflects the local structural asymmetry around the iron-binding sites. The developed experimental approach can probe the relationship between the long-range outer-sphere chemical interaction of a biological redox-active center and the chemical reactivity of the protein.

The biological [2Fe-2S] proteins take on versatile physiological functions in all three domains of life, and in some cases have been implicated as a target for pharmaceutical, bioengineering, and medical applications. In this work, the electron spin transfer onto the metal ligands in all three major classes from this [2Fe-2S](His)n(Cys)4-n (n=0,1,2) protein family is explored by two-dimensional pulsed EPR spectroscopy. This figure illustrates the maps of the long-distance spin transfer onto the ligands of the reduced [2Fe-2S](His)n(Cys)4-n (n=0,1,2) protein family, where the Rieske-type [2Fe-2S](Cys)2(His)2 ferredoxin ARF (n=2, top), the ISC-like [2Fe-2S](Cys)4 ferredoxin FdxB (n=0, middle), and the thermophile mitoNEET-type [2Fe-2S](Cys)3(His)1 protein TthNEET (n=1, bottom) are used as representative models for each cluster type (see Taguchi et al. (2018) Inorg. Chem. 57, 741-746). Positive electron spin density is shown in blue, negative spin density in red, and the spin density is shown in green where the sign is unknown. The volumes of the circles indicate the relative spin populations for the Cys-Cβ and His-Nε atoms. This figure was prepared by Dr. Alexander Taguchi.

Background and Explanation

Dr. Sergei Dikanov and Dr. Toshio Iwasaki are supported from their respective funding agencies, NSF (CHE-1026541) and JSPS (FY2010) in Japan, through the International Collaboration in Chemistry Program. Their collaborative research makes use of state-of-art magnetic resonance-based approaches to study how biological (paramagnetic) metal centers control the power-producing conduits in the cells. Through bi-directional visits to each laboratory, team members are supported for studying new experimental techniques not available in their parent institutions, exchanging scientific ideas, and gaining the experience that comes with living under different cultural atmospheres.


Iwasaki, T., Fukazawa, R., Miyajima-Nakano, Y., Baldansuren, A., Matsushita, S., Lin, M. T., Gennis, R. B., Hasegawa, K., Kumasaka, T., and Dikanov, S. A. (2012) Dissection of hydrogen bond interaction network around an iron-sulfur cluster by site-specific isotope labeling of hyperthermophilic archaeal Rieske-type ferredoxin. J. Am. Chem. Soc. 134, 19731-19738. Pubmed

Lin, M. T., Fukazawa, R., Miyajima-Nakano, Y., Matsushita, S., Choi, S. K., Iwasaki, T., and Gennis, R. B. (2015) Escherichia coli auxotroph host strains for amino acid-selective isotope labeling of recombinant proteins. Methods Enzymol. (Isotope Labeling of Biomolecules - Labeling Methods), 565, 45-66. Pubmed

Taguchi, A. T., Miyajima-Nakano, Y., Fukazawa, R., Lin, M. T., Baldansuren, A., Gennis, R. B., Hasegawa, K., Kumasaka, T., Dikanov, S. A.,* and Iwasaki, T.* (2018) The unpaired electron spin density distribution across reduced [2Fe-2S] cluster ligands by 13Cβ-cysteine labeling. Inorg. Chem. 57, 741-746. Pubmed

Taguchi, A. T.,* Ohmori, D., Dikanov, S. A., and Iwasaki, T.* (2018) g-Tensor directions in the protein structural frame of hyperthermophilic archaeal reduced Rieske-type ferredoxin explored by 13C pulsed electron paramagnetic resonance. Biochemistry 47, 4074-4082. Pubmed

Iwasaki, T.,* Miyajima-Nakano, Y., Fukazawa, R., Lin, M. T., Matsushita, S., Hagiuda, E., Taguchi, A. T., Dikanov, S. A., Oishi, Y., and Gennis, R. B. (2021) Escherichia coli amino acid auxotrophic expression host strains for investigating protein structure-function relationships. J. Biochem. (Review) 169, 387-394. Pubmed    **Selected for journal cover image


The projections of the 13C HYSCORE spectral illustrations onto The Lewis Chessmen in the British Museum, London / Copyright 2018, Toshio Iwasaki. All Rights Reserved.

Research Background

-for researchers-

The metallo-sulfur redox sites, often containing sulfurs from cysteinyl side chains, are the most common in biological redox-active metalloproteins. In particular, modular proteins containing iron-sulfur clusters are widely distributed across all living organisms and have been considered to be of early evolutionary origin. The substitution and/or displacement events at the local metal-binding site(s) in a protein might have greatly enhanced their capabilities of conducting a wide range of unique redox chemistry in biological electron transfer conduits. In addition to their electron transfer roles in respiration and photosynthesis, iron-sulfur proteins are also known to participate in nitrogen fixation, gene regulation, detoxification and environmental sensing, and are suggested to be involved in several human diseases (e.g. Parkinson's disease, Friedreich's ataxia). In all these systems, the interplay of the iron-sulfur cluster core with the surrounding protein is the key to in-depth understanding of the underlying reaction mechanisms.

While X-ray crystallography provides a complementary but rather static picture of the molecular architecture of a protein, high-resolution electron paramagnetic resonance (EPR) techniques, such as pulsed and multifrequency EPR, can potentially provide detailed information about each measurable paramagnetic center therein and its magnetic interplay with the reaction neighborhood in the functional states (Fig. 1). Despite 30 years of research history, there is currently no example of a fully characterized biological iron-sulfur cluster in its paramagnetic ground state via (a) its g-tensor [including principal values and axes within the molecular frame of a protein] and (b) the 3D landscape showing the distribution of unpaired spin density transferred from the cluster over the coordinated/non-coordinated neighboring amino acids in any tractable protein. The plan for our multidisciplinary international collaboration is therefore directed toward this new goal, with innovative features.

Figure 1. Two-dimentional pulsed EPR (also called hyperfine sublevel correlation (HYSCORE)) spectra in 3D presentation of the uniformly 15N-labeled hyperthermostable archaeal Rieske-type ferredoxin (ARF)from Sulfolobus solfataricus (Refs.1,2)(A) and superimposed stacked HYSCORE spectra in the (++) quadrant of 15N-ARF (blue) and 15N-sulredoxin (Refs.3,4)(red) (B), recorded near the gz area. At least two superimposed but well-resolved pairs of the cross-peaks are clearly detected at [2.0; 0.92] MHz (15Np (peptide nitrogen)) and [1.7; 1.2] MHz (15Nepsiron of histidine ligands) with the splittings of 1.1 and 0.5 MHz, respectively, near gz (A). Additional contribution to the 15N ESEEM amplitude in the (++) quadrant of the spectra is evident for 15N-sulredoxin (red) [and other high-potential Rieske [2Fe-2S] proteins; not shown], when the stacked spectra (with zero projection angles) were re-scaled and superimposed after normalizing the relative scales of the cross-peak intensities from two histidine Ndelta ligands in the (+-) quadrant (B) (Ref.1). The same small tau-value (tau = 136 ns; slightly exceeding the dead time of the instrument) was chosen for the measurement of these 15N HYSCORE spectra, which allows the preferable observation of the undistorted lineshape of the cross-peaks as well as the minimization of the suppression effect on the electron spin-echo envelope modulation (ESEEM) amplitudes. Magnetic field, and microwave frequency, respectively: 342.5 mT (15N-ARF) and 344.3 mT (15N-SDX) (near gz), 9.695 GHz.

Our approach is to employ the states-of-art high-resolution pulsed EPR (Fig. 1) and X-ray crystallography (Fig. 2) to study the immediate reaction neighborhood of the paramagnetic centers of our hyperthermostable tractable metalloproteins. Modern EPR techniques probe the magnetic interaction between the electron spin of a paramagnetic cluster and the neighboring nuclear spin of a protein (Fig. 1), and can provide a grand picture of the local spatial and electronic structure relevant to function (specifically for paramagnetic states of the redox centers), not addressed solely from crystallography and not so limited by protein size (problematic in solution NMR studies). We will investigate the three-dimensional landscape of the spin density distribution around the key amino acid residues having the major influence on the electronic structure of the reduced [2Fe-2S](His)2(Cys)2 cluster in our hyperthermophilic tractable Rieske protein, by combined efforts of X-ray crystallography, pulsed EPR and 15N, 13C, 57Fe labeling. This will be used to analyze the strength of the N-H...S hydrogen bonds in relation to their orientation relative to the g-tensor axes and the atomic coordinates defined by the available high-resolution structure. We will also analyze the precise variations of electronic structure under the influence of the histidine ligand(s) and other neighboring residues in three types of our tractable proteins with a reduced [2Fe-2S](His)n(Cys)4-n cluster in the ligand raw (n=0,1,2) (Fig. 2). We would expect that, in general, our approach using high-resolution structures of thermophile proteins coupled with information from spectroscopy and theoretical calculation can help to resolve ambiguities regarding the mechanical and functional hypotheses of metalloenzymes in the future studies on the chemistry of life.

Figure 2. Comparative structures of (A) FdxB at 1.90 Å resolution (Ref.5), (B) the Thermus thermophilus homolog of mitoNEET at 1.80 Å resolution (Ref.6), and (C) the native sulredoxin from Sulfolobus tokodaii at 1.15 Å resolution, with emphasis on the [2Fe-2S](Cys)4-n(His)n cluster binding site (n=0,1,2) [to be published].

This interdisciplinary research project is based on the intensive collaborative work between the research groups of Dr. Sergei A. Dikanov from University of Illinois, and those of Dr. Toshio Iwasaki from Nippon Medical School (Tokyo, Japan) and Dr. Takashi Kumasaka from JASRI/SPring-8 (Hyogo, Japan). Dr. Dikanov has extensive experience in different advanced EPR techniques including two-dimensional (2D) electron spin-echo envelope modulation (ESEEM), electron nuclear double resonance (ENDOR), high-field EPR spectroscopies and their applications to biological macromolecules. A limited number of laboratories worldwide are specialized in state-of-art 2D ESEEM and ENDOR spectroscopy, which will be broadly used in this proposed study. The group of Dr. Iwasaki focuses on biochemical and biophysical studies of a broad class of proteins containing iron-sulfur clusters, in tight collaboration with Dr. Kumasaka who has been playing a leading role in high-resolution structural determination of their protein crystals by X-ray diffraction using synchrotron radiation. The primary interest in this collaboration is in development of novel methods for more rapid and deeper structural characterization of metalloenzymes, using the state-of-art EPR (ESEEM/ ENDOR) methods, which complements the X-ray crystallography. The first experiments using pulsed EPR and double resonance were done in the 1990's, but these provided limited information. Our new approach, using single-crystal measurements and specific labelling of amino-acid residues is more challenging, but it is exactly what is needed to move onto the next stage: a full electronic description of the biological iron-sulfur clusters. Thus, the mutual interests of groups from the US and Japan ideally match each other, and the international collaboration among them is highly appropriate and strengthens their research opportunities as well as the quality of their scientific results. This project will support active efforts to facilitate the mentorship and the "equal-opportunity" recruitment of graduate students/postdoctoral researchers/junior investigators from the underrepresented groups for the international scientific collaborative experience, providing them with access to equipment and expertise not available in each institution, and the living experience under different cultural atmospheres. Moreover, we plan to translate and interpret our main results in formats understandable and useful for non-scientists in the website.


The recent availability of huge amounts of genomic sequence information in conjunction with high-throughput "-omics" analyses has led to a number of unexpected discoveries of novel metallo-sulfur proteins in a wide range of chemistry functions, such as energy conversion, metabolism, and gene regulation, that drive the biosphere on Earth. The potential relationship between some of these metalloenzymes and human healthcare is also at an interesting stage of development where a main focus is on the structure-function interface. Although there are a number of sophisticated techniques and theories that can address the electronic structure of a biological metallo-sulfur cluster core and related model compounds, few can be applied directly to probe the electronic interplay between a metallo-sulfur center and the immediate protein environment, especially in a massive metalloenzyme complex carrying multiple redox centers. Combined efforts of protein X-ray crystallography and pulsed EPR in conjunction with isotope labeling are ideal approaches for this purpose. The practical benefits of the proposed study arise from the fact that the paramagnetic, polynuclear iron-sulfur intermediates are at the core of many key redox and/or iron-sulfur chemistry based catalytic reactions in biological systems. These are so central to life processes that specific roles can be identified at many levels, from biomedical applications (understanding drug metabolism, mitochondrial iron overload and other related disorders, etc.), agronomy (plant photosynthesis, nitrogen fixation, etc.), or microbial contribution to global cycling of elements (carbon, nitrogen, sulfur, hydrogen, etc.). If achieved the results of this work would have a significant impact on these studies by increasing the quality of research and effectiveness in the application of the modern EPR approaches and by making it possible to open the door for answering the next generation of hitherto intractable research problems.


Model Fe-S proteins used in this work (include links to learn more):

X-band Bruker ELEXSYS E580 spectrometer at UIUC / Courtesy of Dr. Sergei A. Dikanov (UIUC).

Sulredoxin (archaeal Rieske [2Fe-2S](His)2(Cys)2 protein)

ARF  (archaeal Rieske-type [2Fe-2S](His)2(Cys)2 ferredoxin)

TthNEET0026  (thermophile mitoNEET [2Fe-2S](His)1(Cys)3 homolog)

FdxB  (ISC-like [2Fe-2S](Cys)4 ferredoxin from Pseudomonas putida)


Amino acid-selective isotope labeling is an extremely powerful method to elucidate specific contributions of particular residues in the reaction mechanisms and/or folding of a target protein by magnetic resonance (e.g., nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR)) and vibrational (e.g., resonance Raman (RR) and Fourier transform infrared (FTIR)) spectroscopies, often aided by the X-ray crystal structure. These techniques can provide detailed information about protein–protein and protein–ligand interactions and dynamics.

One of the most convenient and cost-effective procedures for selective isotope labeling of proteins is to employ amino acid auxotrophic bacteria as the host strains for the overproduction of target proteins. A wild-type Escherichia coli strain has the ability to synthesize all 20 amino acids, whereas an E. coli auxotroph, having an essential gene involved in the biosynthesis of an amino acid disrupted, requires that particular amino acid for growth. However, no suitable auxotrophic strains are commercially available for high-level expression of the foreign genes coding for metalloenzymes from extremophilic archaea and bacteria, because (i) their high-level expression, e.g., in E. coli, often requires extra copies of tRNA genes for the cognate rare codons and (ii) specific growth conditions must be set for effective overproduction of holoproteins in a form suitable for biophysical studies.

To overcome these problems, we have reported the construction of a set of new C43(DE3) and BL21(DE3) auxotrophic expression host strains of E. coli designed to facilitate the labeling of either membrane proteins or water-soluble proteins with selected amino acid types enriched with stable isotopes such as 2H, 13C and 15N [Methods Enzymol. 565, 45-66 (2015); J. Biochem. (Review) 169, 387-394 (2021)]. They have been deposited to public strain banks (available through either LinkIconAddgene, USA, or LinkIconRIKEN BRC, Japan <https://dnaconda.riken.jp/search/depositor/dep006963.html>). The use of a suitable auxotrophic expression strain with the corresponding input isotope labeled amino acid(s) in the growth medium ensures high levels of efficiency as well as selectivity in stable isotope labeling, and is expected to solve many selective labeling problems.

Use the links to learn more about the group's research and publications on this project.


inserted by FC2 system