5YGQ

Kinetic and structural insight into a role of the re-face Tyr328 residue of the homodimer type ferredoxin-NADP+oxidoreductase from Rhodopseudomonas palustris in the reaction with NADP+/NADPH.

(2019) Biochim Biophys Acta Bioenerg : 148140-148140

• PubMed: 31838096 Search on PubMed
• DOI: https://doi.org/10.1016/j.bbabio.2019.148140
• Primary Citation of Related Structures:  
  5YGQ


• PubMed Abstract: 
  

  Among the thioredoxin reductase-type ferredoxin-NAD(P) ⁺ oxidoreductase (FNR) family, FNR from photosynthetic purple non‑sulfur bacterium Rhodopseudomonas palustris (RpFNR) is distinctive because the predicted residue on the re-face of the isoalloxazine ring portion of the FAD prosthetic group is a tyrosine. Here, we report the crystal structure of wild type RpFNR and kinetic analyses of the reaction of wild type, and Y328F, Y328H and Y328S mutants with NADP ⁺ /NADPH using steady state and pre-steady state kinetic approaches. The obtained crystal structure of wild type RpFNR confirmed the presence of Tyr328 on the re-face of the isoalloxazine ring of the FAD prosthetic group through the unique hydrogen bonding of its hydroxyl group. In the steady state assays, the substitution results in the decrease of K _d for NADP ⁺ and K _M for NADPH in the diaphorase assay; however, the k _cat values also decreased significantly. In the stopped-flow spectrophotometry, mixing oxidized RpFNRs with NADPH and reduced RpFNRs with NADP ⁺ resulted in rapid charge transfer complex formation followed by hydride transfer. The observed rate constants for the hydride transfer in both directions were comparable (>400 s ^-1 ). The substitution did not drastically affect the rate of hydride transfer, but substantially slowed down the subsequent release and re-association of NADP ⁺ /NADPH in both directions. The obtained results suggest that Tyr328 stabilizes the stacking of C-terminal residues on the isoalloxazine ring portion of the FAD prosthetic group, which impedes the access of NADP ⁺ /NADPH on the isoalloxazine ring portions, in turn, enhancing the release of the NADP ⁺ /NADPH and/or reaction with electron transfer proteins.

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Organizational Affiliation: 

Division of Material Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa, Ishikawa 920-1192, Japan. Electronic address: dseo@se.kanazawa-u.ac.jp.