Effect of ferrihaem binding on the electrophoretic mobility of HmuY.

<p>The presence of haem in the complex with HmuY is confirmed by haem-peroxidise activity (bottom panel).</p

Spectrum showing the ferrihaem-HmuY complex (red line) formed after 24 h co-incubation of oxyhaemoglobin with HRgpA and HmuY.

<p>The oxyhaemoglobin and HmuY concentrations were both 16 µM, whilst HRgpA was at 0.4 µM.</p

Spectroscopic demonstration of the formation of the HmuY-ferrihaem complex following interaction with immobilised methaemoglobin.

<p>Methaemoglobin-agarose (16 µM with respect to haemoglobin subunit) was incubated with an equimolar amount of HmuY and at various time periods the agarose beads sedimented by centrifugation and the spectra of the supernatant recorded. The spectrum denoted c represents the small amount of methaemoglobin (∼0.25% of total) spontaneously released from the control methaemoglobin-agarose beads after 6 h in the absence of HmuY. The incubations were carried out at 37°C. See text for details.</p

Formation of the ferrihaem-HmuY complex during incubation of human methaemalbumin with HmuY.

<p>Methaemalbumin (16 µM) was incubated with an equimolar amount of HmuY at 37°C. Arrows denote changes in the spectra with time at the indicated wavelengths.</p

Demonstration HmuY-ferrihaem complex formation after exposure of HRgpA-induced methaemoglobin to HmuY.

<p>Before exposure, black line; 24 h after exposure, red line. For clarity, only the initial and final spectra are shown. HRgpA and HmuY were used at 0.4 and 16 µM, respectively. Starting concentration of haemoglobin was 16 µM (with respect to haemoglobin subunit) and comprised 77% methaemoglobin.</p

SDS-PAGE showing the formation of the HMuY-haem complex after incubation with human methaemalbumin.

<p>Methaemalbumin (16 µM) was with either 16 µM HmuY (track 1) or alone (track 2) for 5 h at 37°C, electrophoresed under non-reducing and then stained for the presence of haem with TMB/H₂O₂ before counterstaining for protein with CBB. Note the depletion of TMB/H₂O₂ staining for haem of the methaemalbum band in track 1 after exposure to HmuY.</p

HmuY-haem complex formation during the co-incubation of oxyhaemoglobin with both HmuY and Kgp.

<p>HmuY and oxyhaemoglobin (both at 16 µM) were incubated at 37°C with Kgp (0.2 µM) and sampled periodically, and subjected to native PAGE. Gel tracks were loaded with ∼12 µg total protein.</p

Formation of the HmuY-ferrihaemcomplex during incubation of methaemoglobin-agarose in the presence of human serum albumin.

<p>HmuY, albumin and methaemoglobin were each present at 16 µM (with the haemoglobin on a subunit basis). Panel A, methaemoglobin-agarose co-incubated with both HmuY and albumin. Panel B, methaemoglobin plus albumin only. The experimental protocol was the same as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017182#pone-0017182-g004" target="_blank">Fig. 4</a>, and the incubations were carried out at 37°C. The spectra are background corrected to take account of the small amount of methaemoglobin released at each time period from the haemoglobin agarose during incubation.</p

Formation of the ferrohaem-HmuY complex during incubation of HmuY with deoxyhaemoglobin-agarose.

<p>Haemoglobin-agarose was deoxygenated with sodium dithionite and maintained anaerobically during the reaction with HmuY. Concentration of haemoglobin was 16 µM (with respect to haemoglobin subunit) as was HmuY. See text for details. The absorbance below 375 nm is due to the presence of dithionite. A small amount of deoxyhaemoglobin (∼0.1% of the total) was released from the control deoxyhaemoglobin-agarose after 40 min incubation in the absence of HmuY.</p

Formation of the HmuY-ferrihaem (A) and HmuY-ferrohaem (B) complexes during reaction of HmuY with iron protoporphyrin IX.

<p>HmuY (16 µM) was reacted with an equimolar amount of iron protoporphyrin IX. In (B) the ferrohaem species was generated by inclusion of 10 mM Na₂S₂O₄ in the buffer. The steep drop in absorbance below 375 nm is due to subtraction of the reference background spectrum of the dithionite-containing buffer.</p