Comparison of Soret shift with activity

The relationship between Soret shift in FeO2 intermediate and activity remain very

complicated. We were interested in the relationship between the wavelength maxima of Soret and oxygen reduction activity as a) FeO2 is the only intermediate we have

observed, irrespective of any mutant, during catalytic two or four electron reduction of O2

and naturally this intermediate eventually decomposes to produce water/ROS and met- form of the protein, b) spectroscopic feature of this intermediate showed distinguishable difference in F33Y CuBMb and G65Y CuBMb mutants from WTswMb which again shows

very different product distribution of O2 reduction from former. Here we tried to find

whether there is a linear relationship between Soret shift of FeO2 intermediate with

decrease of ROS or increase of water production. This kind of study is expected to be very complicated as incorporation of one residue sometime brings a lot of conformational change in neighboring group, solvent network and can be characterized only by using multiple probes. This initial study showed in single mutation Soret shift related more with decrease of ROS production compared to increase in water formation (Table 8.1). L29H Mb shows maximum Soret shift (6 nm) among single mutants. Later produces around 5 times less ROS compared to WTswMb however no improvement in water production was observed. On the other hand F43H, among all single mutation, showed maximum increase in water production from WTswMb but only 2 nm Soret shift was observed in FeO2 intermediate compared to WTswMb. F33Y Mb doesn’t show any blue Soret shift

compared to WTswMb and also shows very similar activity as WTswMb. Whereas G65Y Mb shows only 1 nm Soret shift which is also consistent with little change of product

136

distribution (10 % more water production from WT) from WTswMb. All double mutants, except L29H/F33Y Mb, showed around 30-40% more water production compared to WTswMb and also shows 4 nm Soret shift. Only L29H/F33Y Mb shows similar product distribution as WTswMb though 4 nm Soret shift was observed. The probable reasons for this contradictory behavior of L29H/F33Y were already mentioned previously (in role of tyrosines section). Seven out eight mutants (except L29H/F33Y), described in this manuscript, showed a strong relationship between blue Soret shift and increase in water production or decrease in ROS production. However, the exact nature of this relationship is yet to achieve. This manuscript can be considered as first part of investigation where we described the role individual mutant only on activity. We are working towards getting the crystal structure of important mutants in their oxy form to understand the orientation of water molecule around the distal pocket which could have potential role in activity and Soret shift at the same time.

Table 8.1. Relationship between Soret shift and ROS production in single mutants.

8.4. Conclusion

Our data strongly supports strong protonation from multiple directions on oxy myoglobin is required for four electron reduction of O2 to water in the absence of redox active metal.

A protonation on oxy can weaken the O-O bond to such an extent where it can accept two more electron in the same time scale of electron transfer and complete the four electron reduction of O2. Our data also indicates the novel complete O2 reduction activity

of CuBMb mutants is a combinatorial effect of all three mutations (two histidine and one

tyrosine) i.e. no one or two mutations are sufficient to achieve the activity what comes

Protein Soret λmax (nm) Shift (nm)a ROS production (μM s-1) Relative ROSb (%)

WT Mb 417 0 0.9±0.1 100±17 F33Y Mb 418 -1 0.5±0.04 59±7 G65Y Mb 416 1 0.6±0.1 64±11 F43H Mb 415 2 0.8±0.2 87±23 L29H Mb 411 6 0.3±0.1 28±8 a _{shift Soret λ}

max with respect to WT Mb, b

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after third mutation in each case. However the role of single histidine in increase in water production or decrease in ROS production is clear. Result of this work indicates Phe43His mutation brings the four electon O2 reduction activity in myoglobin which get enhanced in

the presence of other mutation whereas Leu29His demolish the ROS production activity of WtswMb. Crystal structure of F33Y CuBMb, published in our previous work 26),

indicates there are two more water molecules at the active site (total three) compared to wild type myoglobin (which has only one). It is possible these residues play a role to bring more water molecules to make strong hydrogen bonding network at the active site of CuBMb mutants which eventually helps strong protonation on FeO2 hence the O-O bond

cleavage. The potential role of hydrogen bonding and solvent network in O-O bond cleavage in native HCOs has been indicated by many theoretical and computational works before 52,53

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