Investigation of Cyc₁ protein structure stability after H53I mutation using computational approaches to improve redox potential

Highlights

• Cyc₁ is a protein in the respiratory chain of the A.f bacterium that has a role in electron transportation.

• In the H53I mutation, the distance between H2O 2030, 2033 and the water molecule 76 (between Cyc₁ and CoxB) is reduced.

• The hydrogen bond between Cyc₁ and CoxB is strengthened and the electron transfer rate between them is increased.

• Increasing the active site flexibility leads to an increase in E₀ at the mutation point and improved electron transfer.

Abstract

Acidithiobacillus ferrooxidans (Af) is an acidophilic bacterium that grows in rigid surroundings and gets its own energy from the oxidation of Fe²⁺ to Fe³⁺. These bacteria are involved in the bioleaching process. Cyc₁ is a periplasmic protein with a crucial role in electron transportation in the respiratory chain. His53 of the Cyc₁ protein, involved in electron transfer to CoxB, was selected for mutation and bioinformatics studies. His53 was substituted by Ile using PyMol software. Molecular dynamics simulations were performed for wild and mutant types of Cyc₁ protein. The conformational changes of mutated protein were studied by analyzing RMSD, RMSF, SASA, Rg, H Bond, and DSSP. The results of the RMSF analysis indicated an increase in the flexibility of the ligand in the mutant. Finally, active site instability leads to an increase in the value of E₀ at the mutation point and improving electron transfer. On the other, His53 in Cyc₁ is interconnected to Glu126 in CoxB through the water molecule (W76) and hydrogen bonding. In the H53I mutation, there was a decrease in the distance between H2O 2030, 2033, and isoleucine 53, and subsequently, the distance to the water molecule 76 between the two proteins was reduced and strengthens the hydrogen bond between Cyc₁ and CoxB, finally improves electron transfer and the bioleaching process.

Introduction

Cyc₁ from the Cyc₁ gene is a periplasmic protein within the respiratory chain of Acidithiobacillus ferrooxidans (Af) sp FJ2 bacterium (strain ATCC 23270) that has a key role in the electron transportation involved in the process of iron oxidation [1]. Af is a Gram-negative, acidophilic bacterium which grows well in rigid surroundings and obtains its energy mainly from the aerobic oxidation of insoluble ferrous iron (Fe²⁺) to soluble ferric iron (Fe³⁺) in acidic environments [2,3].

Af has been introduced to be applied in the metal bioleaching process. Bioleaching is the extraction of metals from their minerals using microorganisms. If the ore does not have enough metal concentration to extract applying conventional chemical methods, then bioleaching is a suitable approach [2]. The ability of acidophilic microorganisms to oxidize metal sulfides has many economic benefits such as approximately 10⁵-10⁶ times higher oxidation rate than chemical methods [4]. In fact, the role of bacteria is the continuous production of ionic ferric through converting the iron II into iron III. Ferro Iron (Fe²⁺) oxidation by this bacterium occurs in two upward and downward paths that are in contact with each other in nature. In the downward path, exergonic, electrons are transmitted biologically from Ferro iron to the cytoplasm through a series of electron carriers towards the inner membrane, where they are used to reduce oxygen to the water [3].

The respiratory chain involved in the redox reactions are probably driven by multiple periplasmic proteins [1] expressed by the following genes: Cyc₂, Cyc₁, ORF₁, CoxB, CoxA, CoxC, CoxD, and Rus [5]. The path of the respiratory chain in bacteria is as follows: Fe(II) → Cyc₂ → rusticyanin → Cyc₁ → cytochrome oxidase aa₃ (Cox) → O₂ cytoplasmic [6,7]. Cyc₁ contains two heme groups: heme A and heme B. The former interacts with the Cu atom of RCY, and the latter receives electron and transfers it to CoxB. Analysis of the heme groups within the Cyc₁ structure showed that carboxyl groups of heme A and heme B interact directly with each other which demonstrates a possible path of electron transfer to the next receiver protein [7]. It is smaller compared to other proteins in the electron transfer chain and is located between two membranes in the periplasmic space.

His53 (Cyc₁) interconnected to Glu126 (CoxB) through the water molecule)W76(and formatted the intermolecular hydrogen bonds with distances of ∼2.95–3.0 Å [8]. Mukhopadhyay et al. examined the possibility of H-bond intermediates and electron transfer reactions in T. Ferroxidants. In the current study, the results of their investigations on the mutation of His53 to Ile and its effect on the stronger hydrogen bonds were applied. By converting polar and hydrophilic histidine to non-polar and hydrophobic isoleucine, the aliphatic index increases, the tendency to internal accumulation and stabilization of protein structure is created by forming hydrophobic interactions. The side chains of these amino acids can regulate the water molecules around the protein. The aliphatic index can be considered as a positive factor in increasing the thermostability of globular proteins. The higher this value, the greater the heat tolerance of the protein. And this agrees with the thermophilicity of this bacterium because this bacterium grows optimally at 30 °C and pH = 2 [[9], [10], [11]]. In the previous studies, the effect of the mutation on the electron entry point into the protein (E121A) and in this study, the effect of the mutation on the electron exit point of the Cyc₁ protein (histidine 53) and transfer to the next protein (CoxB) was investigated [12]. In fact, it can be said that this protein has two active sites, one at the point where the electron enters Cyc₁ and the other at the point where the electron leaves Cyc₁ to the next protein (CoxB).

Barrett in 2006 demonstrated that by converting His to Met through mutation, the redox potential increases by 400 mv [13]. Kanbi et al. showed that with the substitution of Leu by Met148, the redox potential increases by 130 mv [14]. Given the above two sentences, the conversion of His to Leu can also enhance the redox potential. Since the branched Ile has a similar structure to His, we selected the conversion of His53 to Ile. Furthermore, The Hot Spot server suggested H53I for mutation. As a result, there is a greater tendency to obtain electrons from heme B and, improve the electron transfer rate.

Farahmand et al. and Jafarpour et al. examined the Acidithiobacillus ferrooxidans sp. FJ2 bacterium. This bacterium was isolated from Ramsar hot spring and used in the bioleaching process [15,16]. The present study focuses on the effect of a novel mutation on the same type of bacteria in the Cyc₁ gene to improve the bioleaching process by bioinformatics approaches.