Structural and functional comparison of hemoglobin Glb2-1 of <i>Lotus japonicus</i> with Glb1-1 and leghemoglobins

The legume Lotus japonicus expresses nine hemoglobins, including leghemoglobins (Lbs), class 1 phytoglobin (Glb1-1), and an unusual phytoglobin (Glb2-1). Quantitative PCR, proteomics, and plant mutant analyses indicated that Glb2-1 is mainly present in nodules without replacing Lb function, but is also in roots and photosynthetic tissues. Comparison of hormonal profiles of the knock-out mutants glb1-1, glb2-1, and glb1-1/2-1 showed that Glb1-1 and Glb2-1 have distinct functions. The increase of salicylic acid in the leaves of glb1-1 revealed a role of Glb1-1 in the defense response, which was corroborated by accumulation of pipecolic acid, a metabolite involved in plant immunity. In contrast, the decrease of bioactive jasmonoyl-isoleucine in glb2-1 was consistent with a role of Glb2-1 in the plant's reproductive stage. The mutants also showed changes in cytokinins, gibberellins, and polyamines, but without clear distinctive patterns. The crystal structure of Glb2-1 was determined to 1.6 Å resolution and compared with those of soybean Lba and Arabidopsis Glb1. In combination with mutant versions of Glb2-1, residues Tyr31, His64, and Cys65 were identified as critical for O2-binding stability. Spectral changes in heme coordination when Tyr31 is substituted for Phe highlights the importance of the residue at the B10 position for Lb and Glb function.