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. 2024 Jan 11;128(1):86-95.
doi: 10.1021/acs.jpcb.3c05996. Epub 2023 Dec 21.

Suitability of Adenosine Derivatives in Improving the Activity and Stability of Cytochrome c under Stress: Insights into the Effect of Phosphate Groups

Pranav Bharadwaj  1   2 , Sachin M Shet  1 , Meena Bisht  2 , Dheeraj Kumar Sarkar  3   4 , Gregory Franklin  2 , Nataraj Sanna Kotrappanavar  1   5 , Dibyendu Mondal  1   2
Affiliations

Suitability of Adenosine Derivatives in Improving the Activity and Stability of Cytochrome c under Stress: Insights into the Effect of Phosphate Groups

Pranav Bharadwaj et al. J Phys Chem B. .

Abstract

It is well known that adenosine and its phosphate derivatives play a crucial role in biological phenomena such as apoptosis and cell signaling and act as the energy currency of the cell. Although their interactions with various proteins and enzymes have been described, the focus of this work is to demonstrate the effect of the phosphate group on the activity and stability of the native heme metalloprotein cytochrome c (Cyt c), which is important from both biological and industrial aspects. In situ and in silico characterizations are used to correlate the relationship between the binding affinity of adenosine and its phosphate groups with unfolding behavior, corresponding peroxidase activities, and stability factors. Interaction of adenosine (ADN), adenosine monophosphate (AMP), adenosine 5'-diphosphate (ADP), and adenosine 5'-triphosphate (ATP) with Cyt c increases peroxidase-like activity by up to 1.8-6.5-fold compared to native Cyt c. This activity is significantly maintained even after multiple stress conditions such as oxidative stress and the presence of a chaotropic agent such as guanidine hydrochloride (GuHCl). With binding affinities on the order of ADN < AMP < ADP < ATP, adenosine derivatives were found to stabilize Cyt c by varying the secondary structural features of the protein. Thus, in addition to being a fundamental study, the current work also proposes a way of stabilizing protein systems to be used for real-time biocatalytic applications.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Peroxidase activity of Cyt c in the presence of (A) ADN, (B) ADP, (C) AMP, and (D) ATP.
Figure 2
Figure 2
UV–vis spectroscopy for characterizing the interaction between (A) ADN, (B) AMP, (C) ADP, and (D) ATP with Cyt c at room temperature.
Figure 3
Figure 3
Characterization of interaction between Cyt c and (A) ADN, (B) AMP, (C) ADP, and (D) ATP using FTIR spectroscopy. (E) Second derivative FTIR spectra of native Cyt c and Cyt c incubated with ADN, AMP, ADP, and ATP.
Figure 4
Figure 4
CD spectra of Cyt c and adenosine systems at (A) near UV and (B) far UV region. (C) Corresponding results of secondary structural changes calculated using DICHROWEB.
Figure 5
Figure 5
ζ-Potential of 10 μM Cyt c in water and in the presence of optimized concentrations of ADN, AMP, ADP, and ATP.
Figure 6
Figure 6
Electrostatic potential surface and proposed high affinity bound poses of adenosine derivatives (ADN, AMP, ADP, and ATP) in Cyt c. The residue region 23–44 of Cyt c is highlighted in orange surface and cartoon representations.
Figure 7
Figure 7
SwissDock results depicting the putative binding sites for (A) ADN, (C) AMP, (E) ADP, and (G) ATP. The protein is shown both in surface and cartoon representation (gray), and the ATP derivatives are shown as stick representation (orange). The top ranked cluster position is denoted by C0. (B, D, F, H) The representative best poses from the C0 cluster predicted by the PLIP program for ADN, AMP, ADP, and ATP, respectively, are shown depicting the H-bond (solid blue line), pi-stacking (dotted green line), and salt bridge (dotted yellow line) interactions. The charge centers are shown in yellow spheres. The overlapping region 23–44 (yellow asterisk) is a common putative docking site for the adenosine derivative with phosphate groups (AMP, ADP, and ATP).
Figure 8
Figure 8
(A) Activity and (B) corresponding UV–vis spectrum of Cyt c under oxidative stress. (C) Activity and (D) UV–vis spectrum of Cyt c in the presence of GuHCl.
See this image and copyright information in PMC

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