Functional and Spectroscopic Characterization of Chlamydomonas reinhardtii Truncated Hemoglobins

Chiara Ciaccio; Francisco Ocaña-Calahorro; Enrica Droghetti; Grazia R Tundo; Emanuel Sanz-Luque; Fabio Polticelli; Paolo Visca; Giulietta Smulevich; Paolo Ascenzi; Massimo Coletta

doi:10.1371/journal.pone.0125005

Functional and Spectroscopic Characterization of Chlamydomonas reinhardtii Truncated Hemoglobins

PLoS One. 2015 May 20;10(5):e0125005. doi: 10.1371/journal.pone.0125005. eCollection 2015.

Affiliations

¹ Department of Clinical Sciences and Translational Medicine, University of Roma Tor Vergata, Roma, Italy; Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, Bari, Italy.
² Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Córdoba, Cordoba, Spain.
³ Department of Chemistry "Ugo Schiff", University of Firenze, Sesto Fiorentino (FI), Italy.
⁴ Department of Sciences, Roma Tre University, Roma, Italy; National Institute of Nuclear Physics, Roma Tre University Section, Roma, Italy.
⁵ Department of Sciences, Roma Tre University, Roma, Italy.
⁶ Interdepartmental Laboratory of Electron Microscopy, Roma Tre University, Roma, Italy.

Abstract

The single-cell green alga Chlamydomonas reinhardtii harbors twelve truncated hemoglobins (Cr-TrHbs). Cr-TrHb1-1 and Cr-TrHb1-8 have been postulated to be parts of the nitrogen assimilation pathway, and of a NO-dependent signaling pathway, respectively. Here, spectroscopic and reactivity properties of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4, all belonging to clsss 1 (previously known as group N or group I), are reported. The ferric form of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 displays a stable 6cLS heme-Fe atom, whereas the hexa-coordination of the ferrous derivative appears less strongly stabilized. Accordingly, kinetics of azide binding to ferric Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are independent of the ligand concentration. Conversely, kinetics of CO or NO2- binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are ligand-dependent at low CO or NO2- concentrations, tending to level off at high ligand concentrations, suggesting the presence of a rate-limiting step. In agreement with the different heme-Fe environments, the pH-dependent kinetics for CO and NO2-binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are characterized by different ligand-linked protonation events. This raises the question of whether the simultaneous presence in C. reinhardtii of multiple TrHb1s may be related to different regulatory roles.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Azides / metabolism
Base Sequence
Chlamydomonas reinhardtii / genetics*
Chlamydomonas reinhardtii / metabolism
DNA Primers / genetics
Hydrogen-Ion Concentration
Iron / metabolism
Kinetics
Models, Molecular*
Molecular Sequence Data
Protein Conformation
Sequence Analysis, DNA
Spectrophotometry
Truncated Hemoglobins / chemistry*
Truncated Hemoglobins / classification
Truncated Hemoglobins / metabolism*

Substances

Azides
DNA Primers
Truncated Hemoglobins
Iron

Grants and funding

Part of this work was funded by MINECO (Ministerio de Economia y Competitividad, Spain, Grant no. BFU2011-29338) with support of the European FEDER program, by the Junta de Andalucía (P08-CVI-04157 and BIO-502) and Plan Propio de la Universidad de Córdoba. F.O-C thanks MINECO for a Formación de Personal Investigador fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.