INDICADORES, RECOGIDA DE DATOS Y CANTIDADES

To understand the dramatic difference in activity between WTswMb and CuBMb mutants,

we followed up the oxygen reduction assay with the UV-Vis spectroscopic studies of both tyrosine containing CuBMb mutants and WTswMb under turnover conditions in the

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presence of excess reductant (TMPD/ascorbate) and excess O2. The spectroscopic

signature of wild type myoglobin in this condition showed a prominent Soret at 417 nm and visible bands at 542 and 580 nm which matched well with previously reported end- on oxy –product with heme iron known as oxy myoglobin (Fe(II)-O2) 34. The spectral

features of CuBMb mutants under similar condition shows very similar spectroscopic

features like wild type myoglobin except one striking difference, which is a clear 6 nm blue Soret shift. F33Y-CuBMb and G65Y-CuBMb display the Soret band maxima at 411 nm,

whereas the visible part of the spectra remains unperturbed. This distinguishable Soret

Figure 8.3. Spectral feature of FeO

2 intermediate of A) single and B) double mutants

compared with WT Mb and triple mutants. Red line in the Fig. indicates position of λ_max of Soret band and the corresponding numbers have been mentioned at left corner of each spectrum. Final spectra of FeO

2 intermediate was collected from reaction of Met form (6

µM) with 1000 eq of ascorbate and 100 eq of TMPD in the presence of excess O₂. The visible part of the traces have been included in supporting information (Fig. S2)

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shift with overall similar feature with wild type myoglobin indicates an identical FeO2

intermediate with a minute modification. Considering the position of ligand O2 in reported

crystal structure of WT Mb 35 _{and distal pocket mutation in Cu}

BMb, one can speculate a

role of H-bonding interactions between iron bound oxy ligand and newly incorporated histidines (29 and 43) behind this minute modification hence the Soret band shift. A similar spectroscopic Fe-O2 intermediate or UV-Vis signature was assigned as first

intermediate during catalytic O2 reduction to water in HCOs which undergoes O-O bond

Figure 8.4. Spectral features of fluoride bound WT Mb, single mutants and F33Y Cu_BMb. Red line in the Fig. indicates position of λ

max of CT band and the corresponding numbers

have been mentioned at left corner of each spectrum. In each trace protein was incubated with X mM potassium fluoride at pH Y for Z mins brfore taking the final spectra.

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cleavage to form Fe(IV)=O intermediate, known as PM5,36. On the contrary wild type oxy

myoglobin generates reactive oxygen species through autooxidation reaction which was demonstrated in our previous work and other literature 32,33_{. However, Cu}

BMb mutants

show totally different product distribution where it mostly generates water (~ 80%) 26

instead of ROS unlike WT Mb. It is also important to mention here that Fe-O2 is the only

intermediate that has been detected spectroscopically during catalytic turnover of CuB

mutants. This prominent difference in spectral features and activity between WT and CuBMb mutants and specially the similarities in activity between later and native HCO

drives us to correlate the novel 4 electron O2 reduction activity of CuBMb mutants with

Soret shift to investigate whether the H-bonding interaction in the Fe-O2 species

manifests itself as difference in O2 reduction activity.

To understand the fundamental of Soret shift in oxy spectra of CuBMb mutants, UV-Visible

spectra were collected for all single and double mutants in the presence of excess reductant and excess O2. Excess O2 was used to nullify or minimize the binding affinity

difference between mutants. In each case, 6 µM protein was incubated with 1000 equiv of ascorbate and 100 eq of TMPD at pH 6 in air saturated solution (~ 258 µM of O2 at 25

ºC) in a sealed cuvette to prevent the air exchange. 0.12 mg/mL of catalase was also added in every reaction mixture to avoid any accumulation of reactive oxygen species, which may form in the presence of excess reductant. Upon incubation of the protein with reductant, a slow conversion of met to oxy and then to deoxy was observed (the deoxy spectra was not included in the figure as we didn’t observe any change between wild type Mb and tyrosine containing CuBMb mutants, data not shown). Though deoxy forms as an

intermediate between met and oxy, we only start observing deoxy when O2 concentration

goes down in cuvette due to formation of ROS or water. The visible rate of this conversion is not identical for different mutants and entirely depends on O2 binding affinity and rate

of O2 consumption for respective mutant. After complete or semi conversion to deoxy in

1 hr, O2 was bubbled into solution and the formation of oxy-bound protein was monitored.

The typical spectroscopic signatures of met (Ferric), deoxy (ferrous) and oxy swMb are obtained around {408, 503, 623 nm}, {434, 556 nm} and {417, 541, 583 nm} respectively. A comparison of UV-visible signature between single and double mutants has been illustrated in Fig. 8.3. A clear indication of Soret shift towards blue was observed for oxy

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spectra as we increase the number of histidine at the distal pocket. However the position and the nature of visible region remained unaffected. The Soret of single mutation F43H Mb remains in between 414-415 nm whereas that of L29H Mb is observed at 411 nm. On the other hand F33Y Mb indicates almost no Soret shift compared to wild type Mb, 417- 418 nm, which is consistent with O2 reduction activity of the same. However the other

tyrosine single mutant i.e. G65Y Mb showed very small shift compared to WT Mb i.e. 416 nm. This indicates Try65 interacts with iron bound oxy more strongly as compared to Tyr33, and this observation is again consistent with activity studies mentioned before. For all the other double mutants F43H/F33Y, L29H/F33Y, F43H/G65Y, L29H/ G65YMb Soret band maxima is observed around 412 -413 nm. This histidine dependent Soret shift at pH 6 could be due to a protonation on oxygen of Fe(III)-O-O- _{because all of our mutation}

were introduced at distal pocket where O2 binds with Fe in an end-on fashion and all new

residues are expected to be protonated at that particular pH. Crystal structure of F33Y CuBMb indicates the presence of two more water molecules at distal pocket compared to

wild type Mb. This suggest new hydrophilic residues like His 43, His 29, Tyr 33 and Tyr65 individually or together stabilize two new water molecules and one can’t rule out the presence of one or both water molecules in single or double mutation mentioned in this work. At this point we don’t have sufficient evidence whether FeO2 interacts with distal

pocket residue directly or through active site water molecules. However, we have observed the magnitude of this blue shift of Soret in oxy spectra in CuBMb mutants

decreases at higher pH like pH 7 for F33Y CuBMb and G65Y-CuBMb (Fig. S1). This pH

effect again reinforces the fact that histidine directly or indirectly interacts with FeO2 as

pKa of histidine normally comes around 6.