2.1.3. YqhD Monomer

When comparing monomers in the 2 µs aggregated trajectory, it can be seen that the oxidation state of the NADP/H cofactor affects the overall monomer conformation significantly. NADP-bound monomer shows an average backbone RMSD of 0.27 ± 0.06 nm with slightly higher variations than NADPH-bound monomer, which has an average RMSD of 0.23 ± 0.05 nm. The distribution of backbone RMSD and Rg values for YqhD monomers is reported in Figure 2. YqhD monomers bound to NADP show higher deviation from the reference crystal structure. The RMSD distribution ranges from 0.13–0.45 nm, with a main peak at 0.31 nm and two additional peaks at 0.23 and 0.17 nm. NADPH-bound YqhD has a higher probability of remaining closer to the reference structure, showing a main peak at 0.19 nm and two additional sharp peaks at 0.25 and 0.15 nm; however, the RMSD distribution is broader, ranging from 0.09–0.47 nm. The Rg value of monomer is 2.07 nm in the crystal structure. NADP-bound monomers show an average Rg of 2.14 ± 0.02 nm, while NADPH-bound monomers have an average Rg of 2.11 ± 0.03 nm. For NADPHbound monomers, Rg values show a broad distribution ranges from 2.04–2.27 nm with three peaks at 2.07, 2.10 and 2.13 nm. NADP-bound monomer exhibits a narrower Rg distribution, ranging from 2.04–2.25 nm, with a main peak at 2.15 nm and a wider shoulder at 2.09 nm. Overall, YqhD monomer bound with reduced NADPH cofactor populates conformations (two peaks) with lower RMSD (<0.2 nm) and Rg (<2.1 nm) values, i.e., close to the crystal structure that has the modified NADPH(OH)<sup>2</sup> cofactor. Thus, some of the structural differences in YqhD monomer relative to the crystal structure can be attributed to the changes in the oxidation states of the cofactor (reflecting relevant, functional states of NADP/H bound to the enzyme); the molecular details are discussed later. **2021**, , x FOR PEER REVIEW 5 of 17

**Figure 2.** The distribution of backbone (**a**) root mean square deviation (RMSD) and (**b**) radius of gyration (Rg) values are shown for the YqhD monomers. The vertical, cyan-colored bar shows the Rg value observed in the crystal structure.

α α α α α α α α It is worthwhile to keep in mind that the RMSD and Rg values are calculated using the crystal structure as a reference, which may not reflect the functional enzyme structure due to its modified cofactor (NADPH(OH)2). Still, the crystal structure gives a good starting point to witness how the enzyme samples different conformations during the binding of oxidized and reduced cofactors. In the simulations, YqhD shows slightly higher structural flexibility, populating diverse conformations with a more compact yet broader, distinctive distribution of Rg values when bound to NADPH (required for reductase function) compared to NADP (released for enzyme turnover). The population of conformations towards lower RMSD

α β

α

and Rg values in the simulations with reduced cofactor indicates the slight change in protein conformation in NADPH binding relative to NADP binding.

**2021**, , x FOR PEER REVIEW 5 of 17

Figure 3a shows the per-residue backbone RMSD for the YqhD domains illustrated in Figure 3b. High deviations are restricted mainly to loop regions and the N- and Cterminus. Both the monomers show significant deviations in the loops that are present at the dimer interface (between monomers) and the interface between domains within each monomer (marked by the cyan colored horizontal bar in Figure 3a). Monomers bound to NADPH show a higher deviation in loop regions α6/α7, α7/α8, α9/α10 and α12/α13 of the metal-binding helical domain then the NADP-bound monomer. α α α α α α α α

α β α **Figure 3.** (**a**) Backbone RMSD per residue for the domains of monomers bound to NADP (black) and NADPH (red) with respect to the crystal structure. Horizontal bars show α-helices (blue), β-sheets (orange), and the residues involved in dimer and monomer interactions (cyan). The cofactor-binding region in Rossmann-type domain and metal-binding regions within the α-helical domain are shown in yellow vertical bars. (**b**) YqhD monomer is in cartoon representation with bound NADPH (green licorice representation) and Zn2+ (red sphere) in the active site. Helices, beta sheets, and C and N terminals are labeled.
