Figure 6. Difference spectra showing HmuY-haem complex formation from HRgpA-methaemoglobin (A) and NaNO₂-induced methaemoglobin (B).

The difference spectra in the visible region show the appearance of the 525 nm and 558 nm bands attributable to formation of the HmuY-ferrihaem complex. Note that the relative extinctions at 527 nm and 558 nm are reversed compared to those in the ferrihaem-HmuY complex shown in Fig. 1, and occur as a result of subtraction of the spectra. Starting concentrations of haemoglobin were 16 µM (haemoglobin subunit basis) and contained 77 and 87% methaemoglobin for the HRgpA- and NaNO₂-treated oxyhaemoglobin, respectively. HRgpA and NaNO₂ concentrations were 0.4 µM and 64 µM, respectively.