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JBIC Journal of Biological Inorganic Chemistry

, Volume 17, Issue 8, pp 1231–1239 | Cite as

Histidine ligand variants of a flavo-diiron protein: effects on structure and activities

  • Han Fang
  • Jonathan D. Caranto
  • Rosalinda Mendoza
  • Alexander B. Taylor
  • P. John Hart
  • Donald M. KurtzJr.Email author
Original Paper

Abstract

Flavo-diiron proteins (FDPs) contain non-heme diiron and proximal flavin mononucleotide (FMN) active sites and function as terminal components of a nitric oxide reductase (NOR) and/or a four-electron dioxygen reductase (O2R). While most FDPs show similar structural, spectroscopic, and redox properties, O2R and NOR activities vary significantly among FDPs. A potential source of this variability is the iron ligation status of a conserved His residue that provides an iron ligand in all known FDP structures but one, where this His residue is rotated away from iron and replaced by a solvent ligand. In order to test the effect of this His ligation status, we changed this ligating His residue (H90) in Thermotoga maritima (Tm) FDP to either Asn or Ala. The wild-type Tm FDP shows significantly higher O2R than NOR activity. Single crystal X-ray crystallography revealed a remarkably conserved diiron site structure in the H90N and −A variants, differing mainly by either Asn or solvent coordination, respectively, in place of H90. The steady-state activities were minimally affected by the H90 substitutions, remaining significantly higher for O2R versus NOR. The pre-steady-state kinetics of the fully reduced FDP with O2 were also minimally affected by the H90 substitutions. The results indicate that the coordination status of this His ligand does not significantly modulate the O2R or NOR activities, and that FDPs can retain these activities when the individual iron centers are differentiated by His ligand substitution. This differentiation may have implications for the O2R and NOR mechanisms of FDPs.

Keywords

Non-heme iron Nitric oxide Dioxygen X-ray crystallography Enzyme kinetics Site-directed mutagenesis 

Abbreviations

FDP

Flavo-diiron protein

FDPox

FMN-diferric FDP

FDPred

FMNH2-diferrous FDP

FMN

Flavin mononucleotide

FMNH2

FMN hydroquinone

NOR

Nitric oxide reductase

O2R

Dioxygen reductase

NROR

NADH:rubredoxin oxidoreductase

Rd

Rubredoxin

ROO

Rubredoxin:oxygen oxidoreductase

PDB

Protein Data Bank

Tm

Thermotoga maritima

MOPS

3-(N-morpholino)propanesulfonic acid

Notes

Acknowledgments

This work was supported by grants from National Institute of Health (RO1 GM040388 to D.M.K. Jr.) and the Robert A. Welch Foundation (AQ-1399 to P. J. H.). Support for the X-ray Crystallography Core Laboratory by the University of Texas Health Sciences Center at San Antonio Executive Research Committee and the Cancer Therapy & Research Center is gratefully acknowledged. We thank Professor Heather Shipley for access to and assistance with the inductively coupled plasma mass spectrometer.

Supplementary material

775_2012_938_MOESM1_ESM.pdf (6.7 mb)
Supplementary material 1 (PDF 6905 kb)

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Copyright information

© SBIC 2012

Authors and Affiliations

  • Han Fang
    • 1
  • Jonathan D. Caranto
    • 1
  • Rosalinda Mendoza
    • 1
  • Alexander B. Taylor
    • 2
  • P. John Hart
    • 2
    • 3
  • Donald M. KurtzJr.
    • 1
    Email author
  1. 1.Department of ChemistryUniversity of Texas at San AntonioSan AntonioUSA
  2. 2.Department of BiochemistryUniversity of Texas Health Science CenterSan AntonioUSA
  3. 3.Department of Veterans Affairs, Geriatric Research, Education, and Clinical CenterSouth Texas Veterans Health Care SystemSan AntonioUSA

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