Re-engineering of a Haem protein : a spectroscopic and functional analysis of Leghaemoglobin

Site directed mutagenic replacement, of key active site residues, in recombinant leghaemoglobin (rLb) has been performed to probe stmcture/function relationships in this and other haem proteins. The role of the active site residues, in nicotinate binding was investigated. Replacement of the tyrosine 30 and histidine 61 residues with alanine, essentially removing the hydrogen-bond stabilisation to the haem-bound nicotinate, resulted in a 20-fold decrease in affinity for nicotinate. A pH-dependent nicotinate binding study was also conducted on rLb (pKa of 5.2) and Glu63Leu (pH-independent), using electronic (through determination of KdS) and NMR spectroscopy, and identified glutamate 63 as the residue regulating nicotinate binding under acidic conditions. A His61Ala variant was constructed to determine the origin of the low-spin haem species evident in leghaemoglobin. The absence of the low-spin signals in the variant, as determined using a range of spectroscopic techniques, confirmed the origin of the low-spin haem species as arising from coordination of the mobile His61 residue to the haem. The mobility of the His61 residue was further exploited to incorporate new haem axial ligation into rLb. Spectroscopic analysis (using electronic absorption, MCD and EPR spectroscopy) of the His61Tyr and His61Lys variants confirmed haem ligation of the new amino acids and revealed similar spectroscopic characteristics to other haem proteins containing similar haem ligation. Characterisation of the His61Arg variant revealed bis- nitrogenous haem ligation, however, only a tentative assignment for the haem axial ligation could be made. New reactivity was incorporated by replacement of His61 with alanine. The His61Ala variant exhibited haem oxygenase reactivity, as evidenced (using a combination of electronic absorption, HPLC and MS techniques) by the degradation of haem to Fe(III)-biliverdin, via the Fe(II)-02 and unstable verdohaem intermediates, in the presence of a reducing agent (ascorbate). However, the haem degradation of His61Ala proceeded with non- regiospecificity, in contrast to the a-regiospecificity observed for HO and Mb.