Structural basis for hydration dynamics in radical stabilization of bilin reductase mutants

Amanda C. Kohler, David D. Gae, Michael A. Richley, Stefan Stoll, Alexander Gunn, Sunghyuk Lim, Shelley S. Martin, Tzanko I. Doukov, R. David Britt, James B. Ames, J. Clark Lagarias, Andrew J Fisher

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Heme-derived linear tetrapyrroles (phytobilins) in phycobiliproteins and phytochromes perform critical light-harvesting and light-sensing roles in oxygenic photosynthetic organisms. A key enzyme in their biogenesis, phycocyanobilin:ferredoxin oxidoreductase (PcyA), catalyzes the overall four-electron reduction of biliverdin IXα to phycocyanobilin - the common chromophore precursor for both classes of biliproteins. This interconversion occurs via semireduced bilin radical intermediates that are profoundly stabilized by selected mutations of two critical catalytic residues, Asp105 and His88. To understand the structural basis for this stabilization and to gain insight into the overall catalytic mechanism, we report the high-resolution crystal structures of substrate-loaded Asp105Asn and His88Gln mutants of Synechocystis sp. PCC 6803 PcyA in the initial oxidized and one-electron reduced radical states. Unlike wild-type PcyA, both mutants possess a bilin-interacting axial water molecule that is ejected from the active site upon formation of the enzyme-bound neutral radical complex. Structural studies of both mutants also show that the side chain of Glu76 is unfavorably located for D-ring vinyl reduction. On the basis of these structures and companion 15N- 1H long-range HMQC NMR analyses to assess the protonation state of histidine residues, we propose a new mechanistic scheme for PcyA-mediated reduction of both vinyl groups of biliverdin wherein an axial water molecule, which prematurely binds and ejects from both mutants upon one electron reduction, is required for catalytic turnover of the semireduced state.

Original languageEnglish (US)
Pages (from-to)6206-6218
Number of pages13
JournalBiochemistry
Volume49
Issue number29
DOIs
StatePublished - Jul 27 2010

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Bile Pigments
Ferredoxins
Hydration
Oxidoreductases
Stabilization
Biliverdine
Electrons
Phycobiliproteins
Tetrapyrroles
Phytochrome
Synechocystis
Light
Molecules
Water
Protonation
Enzymes
Chromophores
Heme
Histidine
Catalytic Domain

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structural basis for hydration dynamics in radical stabilization of bilin reductase mutants. / Kohler, Amanda C.; Gae, David D.; Richley, Michael A.; Stoll, Stefan; Gunn, Alexander; Lim, Sunghyuk; Martin, Shelley S.; Doukov, Tzanko I.; Britt, R. David; Ames, James B.; Lagarias, J. Clark; Fisher, Andrew J.

In: Biochemistry, Vol. 49, No. 29, 27.07.2010, p. 6206-6218.

Research output: Contribution to journalArticle

Kohler, AC, Gae, DD, Richley, MA, Stoll, S, Gunn, A, Lim, S, Martin, SS, Doukov, TI, Britt, RD, Ames, JB, Lagarias, JC & Fisher, AJ 2010, 'Structural basis for hydration dynamics in radical stabilization of bilin reductase mutants', Biochemistry, vol. 49, no. 29, pp. 6206-6218. https://doi.org/10.1021/bi100728q
Kohler AC, Gae DD, Richley MA, Stoll S, Gunn A, Lim S et al. Structural basis for hydration dynamics in radical stabilization of bilin reductase mutants. Biochemistry. 2010 Jul 27;49(29):6206-6218. https://doi.org/10.1021/bi100728q
Kohler, Amanda C. ; Gae, David D. ; Richley, Michael A. ; Stoll, Stefan ; Gunn, Alexander ; Lim, Sunghyuk ; Martin, Shelley S. ; Doukov, Tzanko I. ; Britt, R. David ; Ames, James B. ; Lagarias, J. Clark ; Fisher, Andrew J. / Structural basis for hydration dynamics in radical stabilization of bilin reductase mutants. In: Biochemistry. 2010 ; Vol. 49, No. 29. pp. 6206-6218.
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T1 - Structural basis for hydration dynamics in radical stabilization of bilin reductase mutants

AU - Kohler, Amanda C.

AU - Gae, David D.

AU - Richley, Michael A.

AU - Stoll, Stefan

AU - Gunn, Alexander

AU - Lim, Sunghyuk

AU - Martin, Shelley S.

AU - Doukov, Tzanko I.

AU - Britt, R. David

AU - Ames, James B.

AU - Lagarias, J. Clark

AU - Fisher, Andrew J

PY - 2010/7/27

Y1 - 2010/7/27

N2 - Heme-derived linear tetrapyrroles (phytobilins) in phycobiliproteins and phytochromes perform critical light-harvesting and light-sensing roles in oxygenic photosynthetic organisms. A key enzyme in their biogenesis, phycocyanobilin:ferredoxin oxidoreductase (PcyA), catalyzes the overall four-electron reduction of biliverdin IXα to phycocyanobilin - the common chromophore precursor for both classes of biliproteins. This interconversion occurs via semireduced bilin radical intermediates that are profoundly stabilized by selected mutations of two critical catalytic residues, Asp105 and His88. To understand the structural basis for this stabilization and to gain insight into the overall catalytic mechanism, we report the high-resolution crystal structures of substrate-loaded Asp105Asn and His88Gln mutants of Synechocystis sp. PCC 6803 PcyA in the initial oxidized and one-electron reduced radical states. Unlike wild-type PcyA, both mutants possess a bilin-interacting axial water molecule that is ejected from the active site upon formation of the enzyme-bound neutral radical complex. Structural studies of both mutants also show that the side chain of Glu76 is unfavorably located for D-ring vinyl reduction. On the basis of these structures and companion 15N- 1H long-range HMQC NMR analyses to assess the protonation state of histidine residues, we propose a new mechanistic scheme for PcyA-mediated reduction of both vinyl groups of biliverdin wherein an axial water molecule, which prematurely binds and ejects from both mutants upon one electron reduction, is required for catalytic turnover of the semireduced state.

AB - Heme-derived linear tetrapyrroles (phytobilins) in phycobiliproteins and phytochromes perform critical light-harvesting and light-sensing roles in oxygenic photosynthetic organisms. A key enzyme in their biogenesis, phycocyanobilin:ferredoxin oxidoreductase (PcyA), catalyzes the overall four-electron reduction of biliverdin IXα to phycocyanobilin - the common chromophore precursor for both classes of biliproteins. This interconversion occurs via semireduced bilin radical intermediates that are profoundly stabilized by selected mutations of two critical catalytic residues, Asp105 and His88. To understand the structural basis for this stabilization and to gain insight into the overall catalytic mechanism, we report the high-resolution crystal structures of substrate-loaded Asp105Asn and His88Gln mutants of Synechocystis sp. PCC 6803 PcyA in the initial oxidized and one-electron reduced radical states. Unlike wild-type PcyA, both mutants possess a bilin-interacting axial water molecule that is ejected from the active site upon formation of the enzyme-bound neutral radical complex. Structural studies of both mutants also show that the side chain of Glu76 is unfavorably located for D-ring vinyl reduction. On the basis of these structures and companion 15N- 1H long-range HMQC NMR analyses to assess the protonation state of histidine residues, we propose a new mechanistic scheme for PcyA-mediated reduction of both vinyl groups of biliverdin wherein an axial water molecule, which prematurely binds and ejects from both mutants upon one electron reduction, is required for catalytic turnover of the semireduced state.

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