Multiple putative methemoglobin reductases in C. reinhardtii may support enzymatic functions for its multiple hemoglobins

• Published in: International journal of biological macromolecules 2021-02, Vol.171, p.465-479
• Main Authors: Shandilya, Manish, Kumar, Gaurav, Gomkale, Ridhima, Singh, Swati, Khan, Mohd Asim, Kateriya, Suneel, Kundu, Suman
• Format: Article
• Language: English
• Subjects: Algal hemoglobins and reductases; Chemistry Techniques, Analytical; Chlamydomonas reinhardtii - enzymology; Chlamydomonas reinhardtii - genetics; Chlamydomonas reinhardtii - metabolism; Conserved Sequence; Cytochrome-B Reductase - chemistry; Cytochrome-B Reductase - genetics; Cytochrome-B Reductase - isolation & purification; Cytochrome-B Reductase - physiology; Humans; Kinetics; Methemoglobin - metabolism; Methemoglobin reduction; Models, Molecular; Molecular Docking Simulation; NO dioxygenase; Oxidation-Reduction; Oxygenases - metabolism; Plant Proteins - isolation & purification; Plant Proteins - physiology; Protein Conformation; Protein Domains; Recombinant Proteins - metabolism; Sequence Alignment; Sequence Homology, Amino Acid; Static Electricity; Substrate Specificity; Truncated Hemoglobins - genetics; Truncated Hemoglobins - isolation & purification; Truncated Hemoglobins - metabolism
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2021.01.023

The ubiquitous nature of hemoglobins, their presence in multiple forms and low cellular expression in organisms suggests alternative physiological functions of hemoglobins in addition to oxygen transport and storage. Previous research has proposed enzymatic function of hemoglobins such as nitric oxide dioxygenase, nitrite reductase and hydroxylamine reductase. In all these enzymatic functions, active ferrous form of hemoglobin is converted to ferric form and reconversion of ferric to ferrous through reduction partners is under active investigation. The model alga C. reinhardtii contains multiple globins and is thus expected to have multiple putative methemoglobin reductases to augment the physiological functions of the novel hemoglobins. In this regard, three putative methemoglobin reductases and three algal hemoglobins were characterized. Our results signify that the identified putative methemoglobin reductases can reduce algal methemoglobins in a nonspecific manner under in vitro conditions. Enzyme kinetics of two putative methemoglobin reductases with methemoglobins as substrates and in silico analysis support interaction between the hemoglobins and the two reduction partners as also observed in vitro. Our investigation on algal methemoglobin reductases underpins the valuable chemistry of nitric oxide with the newly discovered hemoglobins to ensure their physiological relevance, with multiple hemoglobins probably necessitating the presence of multiple reductases. •Three algal reductase systems (CrCyb5R, CrMDHAR and CrDLDH) were reported and characterized.•All three reductases can reduce characterized algal hemoglobins (THB1, THB2 and THB9∆).•In vitro results and insilico analysis support the non-specific reduction of hemoglobins by algal reductases.•Enzyme kinetic parameters of CrCyb5R and CrDLDH were calculated under a strict deoxygenated environment.•Methemoglobin reduction by algal reductases strengthen the nitric oxide dioxygenase function of hemoglobins.