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A conformational equilibrium in the nitrogenase MoFe protein with an α-V70I amino acid substitution illuminates the mechanism of H2 formation

Journal Article · · Faraday Discussions
DOI:https://doi.org/10.1039/d2fd00153e· OSTI ID:2340099
 [1];  [2];  [3];  [4];  [5];  [6];  [2];  [1]
  1. Northwestern Univ., Evanston, IL (United States)
  2. Utah State Univ., Logan, UT (United States)
  3. Washington State Univ., Pullman, WA (United States)
  4. Univ. of Oklahoma, Norman, OK (United States)
  5. Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
  6. Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States)
+ Show Author Affiliations
Study of α-V70I-substituted nitrogenase MoFe protein identified Fe6 of FeMo-cofactor (Fe7S9MoC-homocitrate) as a critical N2 binding/reduction site. Freeze-trapping this enzyme during Ar turnover captured the key catalytic intermediate in high occupancy, denoted E4(4H), which has accumulated 4[e—/H+] as two bridging hydrides, Fe2–H–Fe6 and Fe3–H–Fe7, and protons bound to two sulfurs. E4(4H) is poised to bind/reduce N2 as driven by mechanistically-coupled H2 reductive-elimination of the hydrides. This process must compete with ongoing hydride protonation (HP), which releases H2 as the enzyme relaxes to state E2(2H), containing 2[e—/H+] as a hydride and sulfur-bound proton; accumulation of E4(4H) in α-V70I is enhanced by HP suppression. EPR and 95Mo ENDOR spectroscopies now show that resting-state α-V70I enzyme exists in two conformational states, both in solution and as crystallized, one with wild type (WT)-like FeMo-co and one with perturbed FeMo-co. These reflect two conformations of the Ile residue, as visualized in a reanalysis of the X-ray diffraction data of α-V70I and confirmed by computations. EPR measurements show delivery of 2[e—/H+] to the E0 state of the WT MoFe protein and to both α-V70I conformations generating E2(2H) that contains the Fe3–H–Fe7 bridging hydride; accumulation of another 2[e—/H+] generates E4(4H) with Fe2–H–Fe6 as the second hydride. E4(4H) in WT enzyme and a minority α-V70I E4(4H) conformation as visualized by QM/MM computations relax to resting-state through two HP steps that reverse the formation process: HP of Fe2–H–Fe6 followed by slower HP of Fe3–H–Fe7, which leads to transient accumulation of E2(2H) containing Fe3–H–Fe7. In the dominant α-V70I E4(4H) conformation, HP of Fe2–H–Fe6 is passively suppressed by the positioning of the Ile sidechain; slow HP of Fe3–H–Fe7 occurs first and the resulting E2(2H) contains Fe2–H–Fe6. Importantly, it is this HP suppression in E4(4H) that enables α-V70I MoFe to accumulate E4(4H) in high occupancy. In addition, HP suppression in α-V70I E4(4H) kinetically unmasks hydride reductive-elimination without N2-binding, a process that is precluded in WT enzyme.
Research Organization:
Environmental Molecular Sciences Laboratory (EMSL), Richland, WA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
Sponsoring Organization:
National Science Foundation (NSF); USDOE; USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences & Biosciences Division (CSGB); USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF)
Grant/Contract Number:
AC05-76RL01830; SC0010687; SC0010834; SC0018143
OSTI ID:
2340099
Alternate ID(s):
OSTI ID: 1968651
Report Number(s):
PNNL-SA--179825
Journal Information:
Faraday Discussions, Journal Name: Faraday Discussions Vol. 243; ISSN 1359-6640
Publisher:
Royal Society of ChemistryCopyright Statement
Country of Publication:
United States
Language:
English

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Figures / Tables (14)

Fig. 1(p. 2)figure Fig. 1
Fig. 2(p. 3)figure Fig. 2
Fig. 3(p. 5)figure Fig. 3
Fig. 4(p. 6)figure Fig. 4
Fig. 5(p. 7)figure Fig. 5
Fig. 6(p. 8)figure Fig. 6
Fig. 7(p. 11)figure Fig. 7
Scheme 1(p. 11)figure Scheme 1
Table 1(p. 12)table Table 1
Scheme 2(p. 14)figure Scheme 2
Fig. 8(p. 15)figure Fig. 8
Fig. 9(p. 16)figure Fig. 9
Fig. 10(p. 17)figure Fig. 10
Scheme 3(p. 18)figure Scheme 3

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