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Cited by 8Geomagnetism and Paleomagnetism StudiesMagnetic Properties and Synthesis of FerritesMetalloenzymes and iron-sulfur proteins

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The Magnetosome Protein, Mms6 from Magnetospirillum magneticum Strain AMB-1, Is a Lipid-Activated Ferric Reductase

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Singappuli-Arachchige, Dilini, Feng, Shuren, Wang, Lijun, Palo, Pierre E., Shobade, Samuel O., Thomas, Michelle, Nilsen-Hamilton, Marit·2022·International Journal of Molecular Sciences
DOI 10.3390/ijms231810305PMC PMC9499114

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Abstract

Magnetosomes of magnetotactic bacteria consist of magnetic nanocrystals with defined morphologies enclosed in vesicles originated from cytoplasmic membrane invaginations. Although many proteins are involved in creating magnetosomes, a single magnetosome protein, Mms6 from Magnetospirillum magneticum strain AMB-1, can direct the crystallization of magnetite nanoparticles in vitro. The in vivo role of Mms6 in magnetosome formation is debated, and the observation that Mms6 binds Fe3+ more tightly than Fe2+ raises the question of how, in a magnetosome environment dominated by Fe3+, Mms6 promotes the crystallization of magnetite, which contains both Fe3+ and Fe2+. Here we show that Mms6 is a ferric reductase that reduces Fe3+ to Fe2+ using NADH and FAD as electron donor and cofactor, respectively. Reductase activity is elevated when Mms6 is integrated into either liposomes or bicelles. Analysis of Mms6 mutants suggests that the C-terminal domain binds iron and the N-terminal domain contains the catalytic site. Although Mms6 forms multimers that involve C-terminal and N-terminal domain interactions, a fusion protein with ubiquitin remains a monomer and displays reductase activity, which suggests that the catalytic site is fully in the monomer. However, the quaternary structure of Mms6 appears to alter the iron binding characteristics of the C-terminal domain. These results are consistent with a hypothesis that Mms6, a membrane protein, promotes the formation of magnetite in vivo by a mechanism that involves reducing iron.

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Figure 1 Reductase activity of Mms6. Ferric reductase activity was measured as described in Materials and Methods: (A) the reduction of 70 µM Fe3+-citrate by 20 µM Mms6, m1Mms6, m2Mms6, and m3Mms6 under aerobic (atmospheric) conditions in the presence of 100 µM each of NADH and FAD in Buffer A; (B) comparison of the Vmax (gold bars) and KM (blue bars) for Mms6 under aerobic (empty bars) and anaerobic (cross-hatched bars) conditions; (C) ferric reductase activity of Mms6 (20 µM) at three Fe3+-citrate concentrations under anaerobic (argon) conditions in 100 µM NADH and 40 µM FAD in 70 mM Tris, 100 mM KCl, pH 7.5 and 1 mM ferrozine; (D) the reduction of 70 µM Fe3+-citrate by 20 µM Mms6, and various mutants under aerobic conditions. The asterisks denote p values of <0.05 (*) and <0.001 (***) in paired comparisons with Mms6.
Figure 1 Reductase activity of Mms6. Ferric reductase activity was measured as described in Materials and Methods: (A) the reduction of 70 µM Fe3+-citrate by 20 µM Mms6, m1Mms6, m2Mms6, and m3Mms6 under aerobic (atmospheric) conditions in the presence of 100 µM each of NADH and FAD in Buffer A; (B) comparison of the Vmax (gold bars) and KM (blue bars) for Mms6 under aerobic (empty bars) and anaerobic (cross-hatched bars) conditions; (C) ferric reductase activity of Mms6 (20 µM) at three Fe3+-citrate concentrations under anaerobic (argon) conditions in 100 µM NADH and 40 µM FAD in 70 mM Tris, 100 mM KCl, pH 7.5 and 1 mM ferrozine; (D) the reduction of 70 µM Fe3+-citrate by 20 µM Mms6, and various mutants under aerobic conditions. The asterisks denote p values of <0.05 (*) and <0.001 (***) in paired comparisons with Mms6.CC BY · PubMed Central
Figure 2 Mms6 ferric reductase specificity for cofactor and electron donor: (A) Mms6 (left) and C21Mms6 (right) were tested with 100 µM NADH or NADPH as electron donors combined with either 100 µM FAD or FMN as cofactors in 20 mM Tris, 100 mM KCl, 1 mM ferrozine, and 100 mM ferric citrate, pH 7.5. Reaction mixes run in parallel without protein were used as the background to subtract from the results of incubation with 20 µM Mms6 with the combinations of cofactor and electron donor shown; (B) reductase activity (initial velocity) was determined at a range of Fe3+-citrate concentrations to determine the KM ± standard error for the reductase. Shown is the compiled data from 9 independent experiments involving 6 different protein preparations (r2 = 0.80, error of the estimate = 0.16); (C) the Kd of Mms6 was determined under aerobic conditions at pH 7.5 and pH 7 for ferric citrate and ferric chloride, respectively, and anaerobic conditions at pH 7 for ferric chloride. The ferric citrate binding isotherm was created from the averaged results of 11 independent experiments (duplicates per experiment) with 2 assessed by intrinsic fluorescence and the remainder by 55Fe binding with filter capture. In total, 5 different protein preparations were tested. The aerobic and anaerobic ferric chloride binding isotherms are each the average of two independent experiments with one (anaerobic) or two (aerobic) protein preparations and assessed by 55Fe binding with filter capture. On each plot, the estimated Kd ± standard error is shown. Goodness of fit: Fe-citrate, aerobic (r2 = 0.97, error of the estimate = 0.061), FeCl3, aerobic (r2 = 0.99, error of the estimate = 0.057), and FeCl3, anaerobic (r2 = 0.98, error of the estimate = 0.056).
Figure 2 Mms6 ferric reductase specificity for cofactor and electron donor: (A) Mms6 (left) and C21Mms6 (right) were tested with 100 µM NADH or NADPH as electron donors combined with either 100 µM FAD or FMN as cofactors in 20 mM Tris, 100 mM KCl, 1 mM ferrozine, and 100 mM ferric citrate, pH 7.5. Reaction mixes run in parallel without protein were used as the background to subtract from the results of incubation with 20 µM Mms6 with the combinations of cofactor and electron donor shown; (B) reductase activity (initial velocity) was determined at a range of Fe3+-citrate concentrations to determine the KM ± standard error for the reductase. Shown is the compiled data from 9 independent experiments involving 6 different protein preparations (r2 = 0.80, error of the estimate = 0.16); (C) the Kd of Mms6 was determined under aerobic conditions at pH 7.5 and pH 7 for ferric citrate and ferric chloride, respectively, and anaerobic conditions at pH 7 for ferric chloride. The ferric citrate binding isotherm was created from the averaged results of 11 independent experiments (duplicates per experiment) with 2 assessed by intrinsic fluorescence and the remainder by 55Fe binding with filter capture. In total, 5 different protein preparations were tested. The aerobic and anaerobic ferric chloride binding isotherms are each the average of two independent experiments with one (anaerobic) or two (aerobic) protein preparations and assessed by 55Fe binding with filter capture. On each plot, the estimated Kd ± standard error is shown. Goodness of fit: Fe-citrate, aerobic (r2 = 0.97, error of the estimate = 0.061), FeCl3, aerobic (r2 = 0.99, error of the estimate = 0.057), and FeCl3, anaerobic (r2 = 0.98, error of the estimate = 0.056).CC BY · PubMed Central
Figure 3 Iron binding and reductase activities are correlated: (A) the ferric reductase activity of each recombinant protein was determined as described in Materials and Methods with 20 µM protein and 100 µM ferric citrate; (B) the iron binding activity of each recombinant protein was determined as described in Materials and Methods with 1 µM protein and 20 µM ferric citrate. Reductase and iron binding activities were normalized to that of the wild-type protein. The dashed line identifies the binding activity of m3Mms6, which has been previously shown to bind Fe3+ nonspecifically [9].
Figure 3 Iron binding and reductase activities are correlated: (A) the ferric reductase activity of each recombinant protein was determined as described in Materials and Methods with 20 µM protein and 100 µM ferric citrate; (B) the iron binding activity of each recombinant protein was determined as described in Materials and Methods with 1 µM protein and 20 µM ferric citrate. Reductase and iron binding activities were normalized to that of the wild-type protein. The dashed line identifies the binding activity of m3Mms6, which has been previously shown to bind Fe3+ nonspecifically [9].CC BY · PubMed Central
Figure 4 Primary and tertiary structural analysis of Mms6: (A) alignment of Mms6 and proteins with related sequence. Primary sequences were obtained from the NIH protein database (http://www.ncbi.nlm.nih.gov/protein, accessed on 12 June 2021) and aligned using the clustalw2 online server. The colored regions indicate identity of sequence with the blue region identifying the GL domain and the red rectangle the conserved tryptophan; (B) the AlphaFold2 predicted structure for Mms6 with the conserved Trp and GL domain identified and the primary sequence below. See Figure S8 for 3D models of Mms6 predicted by other algorithms.
Figure 4 Primary and tertiary structural analysis of Mms6: (A) alignment of Mms6 and proteins with related sequence. Primary sequences were obtained from the NIH protein database (http://www.ncbi.nlm.nih.gov/protein, accessed on 12 June 2021) and aligned using the clustalw2 online server. The colored regions indicate identity of sequence with the blue region identifying the GL domain and the red rectangle the conserved tryptophan; (B) the AlphaFold2 predicted structure for Mms6 with the conserved Trp and GL domain identified and the primary sequence below. See Figure S8 for 3D models of Mms6 predicted by other algorithms.CC BY · PubMed Central
Figure 5 Mms6 interaction with lipids: (A) dynamic light scattering analysis of samples (500 µL in 20 mM Tris, 100 mM KCl, pH 7.5) of 25 mM bicelles, 2 mM liposomes or 0.5% Triton X100 with or without 20 µM Mms6 as stated in the legend on the figure. Size distributions for each sample are shown as a volume percentage; (B) fluorescence spectra were collected (λex = 290 nm) and analyzed as described in Materials and Methods. Smoothed curves are shown for the fluorescence intensity of Mms6 (I) or W119F Mms6 (II). The vertical pink and green bars identify the positions of the maximum fluorescence intensity (±2.5 nm, based on a slit width of 5 nm) for each peak colored in red and green, respectively.
Figure 5 Mms6 interaction with lipids: (A) dynamic light scattering analysis of samples (500 µL in 20 mM Tris, 100 mM KCl, pH 7.5) of 25 mM bicelles, 2 mM liposomes or 0.5% Triton X100 with or without 20 µM Mms6 as stated in the legend on the figure. Size distributions for each sample are shown as a volume percentage; (B) fluorescence spectra were collected (λex = 290 nm) and analyzed as described in Materials and Methods. Smoothed curves are shown for the fluorescence intensity of Mms6 (I) or W119F Mms6 (II). The vertical pink and green bars identify the positions of the maximum fluorescence intensity (±2.5 nm, based on a slit width of 5 nm) for each peak colored in red and green, respectively.CC BY · PubMed Central
Figure 6 Mms6 has higher ferric reductase activity in lipid environments than in the absence of lipid: (A) Mms6, in liposomes of various lipid compositions as shown in the legend in the figure, was assayed in the presence of 100 µM each of NADH and FAD for ferric reductase activity with 0.8 mM ferrozine. The molar ratio of lipid:Mms6 was 100:1. The full chemical names of these lipids are defined in Materials and Methods; (B) Mms6 alone or incorporated into 25 mM q = 1 DMPC/DHPC bicelles was assayed in the presence of 200 µM each of NADH and FAD for reductase activity with 1.6 mM ferrozine; (C) the Vmax of Mms6 and identified mutants while free in solution (green bars) or associated with 12.5 mM bicelles (red bars) were determined in the presence of 100 µM each of NADH and FAD with 0.8 mM ferrozine. In all assays, Mms6 was 20 µM and the buffer was 20 mM Tris, 100 mM KCl, pH 7.5.
Figure 6 Mms6 has higher ferric reductase activity in lipid environments than in the absence of lipid: (A) Mms6, in liposomes of various lipid compositions as shown in the legend in the figure, was assayed in the presence of 100 µM each of NADH and FAD for ferric reductase activity with 0.8 mM ferrozine. The molar ratio of lipid:Mms6 was 100:1. The full chemical names of these lipids are defined in Materials and Methods; (B) Mms6 alone or incorporated into 25 mM q = 1 DMPC/DHPC bicelles was assayed in the presence of 200 µM each of NADH and FAD for reductase activity with 1.6 mM ferrozine; (C) the Vmax of Mms6 and identified mutants while free in solution (green bars) or associated with 12.5 mM bicelles (red bars) were determined in the presence of 100 µM each of NADH and FAD with 0.8 mM ferrozine. In all assays, Mms6 was 20 µM and the buffer was 20 mM Tris, 100 mM KCl, pH 7.5.CC BY · PubMed Central
Figure 7 Monomeric Mms6 is a functional reductase but with altered C-terminal domain specificity for Fe3+: (A) (1) eluted peaks of standards, Mms6 and Ubi-Mms6; (2) standard curve derived from (1) with a vertical green line showing the elution position of Ubi-Mms6, MW: molecular weight, Ve/Vo = elution volume/void volume; (3) gel electrophoretic profile of Ubi-Mms6 prior to loading and in the peak eluted at ~15 mL std: molecular weight standard proteins; input: Ubi-Mms6 sample loaded on the column, peak, sample from the Ubi-Mms6 peak as identified in the A280 profile (A1); (B) time courses of reductase activity in the presence of 350 µM ferric citrate in 20 mM Tris, 100 mM KCl, pH 7.5 with 100 uM each of NADH and FAD with 0.8 mM ferrozine. For each binding isotherm, the data from three independently performed experiments, each normalized to a midpoint value, were averaged, and similarly treated data in the presence of each protein but no ferric citrate was subtracted. The error bars are the square root of the sum of the squares of the errors with and without ferric citrate; (C) the amount of iron bound per µmole of protein in 20 µM 55Ferric citrate was determined as described in Materials and Methods and expressed as a proportion of the amount bound by Mms6. The asterisks denote a p value of <0.05 (*) in a paired comparison with Mms6.
Figure 7 Monomeric Mms6 is a functional reductase but with altered C-terminal domain specificity for Fe3+: (A) (1) eluted peaks of standards, Mms6 and Ubi-Mms6; (2) standard curve derived from (1) with a vertical green line showing the elution position of Ubi-Mms6, MW: molecular weight, Ve/Vo = elution volume/void volume; (3) gel electrophoretic profile of Ubi-Mms6 prior to loading and in the peak eluted at ~15 mL std: molecular weight standard proteins; input: Ubi-Mms6 sample loaded on the column, peak, sample from the Ubi-Mms6 peak as identified in the A280 profile (A1); (B) time courses of reductase activity in the presence of 350 µM ferric citrate in 20 mM Tris, 100 mM KCl, pH 7.5 with 100 uM each of NADH and FAD with 0.8 mM ferrozine. For each binding isotherm, the data from three independently performed experiments, each normalized to a midpoint value, were averaged, and similarly treated data in the presence of each protein but no ferric citrate was subtracted. The error bars are the square root of the sum of the squares of the errors with and without ferric citrate; (C) the amount of iron bound per µmole of protein in 20 µM 55Ferric citrate was determined as described in Materials and Methods and expressed as a proportion of the amount bound by Mms6. The asterisks denote a p value of <0.05 (*) in a paired comparison with Mms6.CC BY · PubMed Central

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Metadata

Authors
Singappuli-Arachchige, D., Feng, S., Wang, L., Palo, P. E., Shobade, S. O., Thomas, M., & Nilsen-Hamilton, M.
Affiliations
  • Ames Laboratory, U.S. Department of Energy, Ames, IA 50011, USA
  • United States Department of Energy (US)
ORCID
Venue
International Journal of Molecular Sciences
Publisher
MDPI AG
Published
2022-09-07
Volume
23
Issue
18
Pages
10305
ISSN
1422-0067 (ISSN-L)
DOI
10.3390/ijms231810305
PMC
PMC9499114
OpenAlex
W4295011505
Cited by
8
References
67
Citations by year
2026: 1 · 2025: 2 · 2024: 4 · 2023: 1
FWCI
0.57
Type
journal article
Language
en
Open access
Licence
Locations
  • International Journal of Molecular Sciences (publishedVersion, cc-by)
  • Preprints.org (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
Funders
  • U.S. Department of Energy, Office of Basic Energy Science, Division of Materials Sciences and Engineering (DE-AC02-07CH11358)
  • Aptalogic Inc. (DE-AC02-07CH11358)
  • U.S. Department of Energy by Iowa State University (DE-AC02-07CH11358)
Topics
  • Physical Sciences › Earth and Planetary Sciences › Geophysics › Geomagnetism and Paleomagnetism Studies
  • Physical Sciences › Materials Science › Materials Chemistry › Magnetic Properties and Synthesis of Ferrites
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Metalloenzymes and iron-sulfur proteins
Keywords
magnetosomes, Magnetospirillum magneticum AMB-1, ferric reductase, magnetite crystallization, magnetotactic bacteria, iron binding, C-terminal domain, protein oligomerization, ubiquitin fusion protein
MeSH terms
Bacterial Proteins, Flavin-Adenine Dinucleotide, Iron, Lipids, Liposomes, Membrane Proteins, NAD, Ubiquitins, FMN Reductase, Magnetospirillum, Ferrosoferric Oxide, Magnetosomes

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