Metallopeptidases (MPs) represent a class of enzymes categorized as proteases. Their function is to catalyze hydrolysis reactions of peptide bonds in proteins and/or peptides, as shown in the Fig. 1 [[1], [2], [3], [4]]. Glutamic acid functions as a general base catalyst by facilitating the removal of a proton from the water bound to the metal, enabling the –O-H group to initiate the attack on the peptide carbonyl group (I). Subsequently, Glu serves as a general acid catalyst by providing a proton to the leaving amine as the tetrahedral intermediate (II), which is bound to the metal and collapses into products (III). In the final stage, the N-terminal product dissociates, and water rebinds to the zinc ion (IV). The C-terminal product remains connected to Glu through salt-bridges interaction [5,6]. These enzymes are characterized by the presence of a divalent metal ion in the active site, which plays an essential role in their catalytic activity [1,2,5,7,8]. The most commonly found metal in the active domain is Zn(II), although Co(II), Mn(II), Ca(II), Ni(II), and Cu(II) can also be present, but less frequently [9]. The metal ion coordination primarily involves three side chains of amino acids with charged residues such as histidine, aspartic acid, glutamic acid, or lysine, in addition to a water molecule serving as the fourth ligand. The water molecule undergoes activation and attacks the peptide bond within the substrate [5,10]. Subsequently, the nucleophilic attack causes the peptide bond to be cleaved, resulting in the substrate being divided into two distinct products [8].

There are several classifications of MPs in families. According to the MEROPS database, which serves as a comprehensive resource for all peptidases, they have been divided into 106 families (from M01 to M106), some of which have been further divided into subfamilies [11,12]. One of the clans is the MA, which includes enzymes containing a zinc ion in their active site. It includes well-studied metalloendopeptidases, thermolysins, and other enzymes derived from eukaryotic and prokaryotic organisms. These enzymes have two histidines coordinating the zinc ion, sourced from the motif His-Glu-X-X-His (HExxH), which is located near the domain's N-terminus [10,13]. The MA clan can be subcategorized further. MA(E), in which the third ligand of the Zn(II) ion is glutamic acid; MA(M), where the third ligand is either histidine or asparagine (HExxHxxGxxH/D). The glycine within this motif plays a structural role as it allows for protein folding, frequently coinciding with the formation of β-turns and α-helices. This turn allows the peptide to bring the zinc ligands into closer proximity, resulting in an optimal special arrangement, which promotes efficient metal coordination [13,14].

Metallopeptidases can also be classified based on the number of metal ions necessary for their catalytic activity: most MPs operate with only one metal ion, like Zn(II)-MPs, while some families depend on two metal ions. Specifically, MPs containing Co(II) or Mn(II) in their active sites necessitate the involvement of two metal ions. Interestingly, there are zinc-dependent families in which two Zn(II) ions act as co-catalysts, whereas all Ni(II)-containing enzymes require only one metal ion [9].

The role of MPs encompasses a wide range of physiological and pathological processes, including tissue remodeling, development, wound healing, immune response, and disease progression [4,10]. MPs found in Streptococcus pneumoniae bacteria are considered one of the virulence factors that help spread infection in the host [15]. These proteases have the ability to break down host organ and tissue basement membranes, as well as extracellular matrix proteins, which assist the pathogenic infection [16,17]. Some of them can cleave human IgA1, which is significant in lung infections and sepsiscases of lung infections and sepsis [3,18]. Because MPS play a critical role in the pathogenesis of many diseases, there is growing interest in their role in human diseases [[19], [20], [21]]. Given the recent success of protease modulators treating diseases, such as regulating blood glucose levels (dipeptidyl peptidase 4) [22], and HIV therapy [23], targeting bacterial MPs essential to virulence, represents a promising therapeutic strategy for the upcoming generation of antibacterial treatments [16,19,20]. The biological role of potential drugs would be based on protease inhibition, which in the case of MPs could be aimed at the catalytically active zinc-binding domain. Therefore, understanding their functions, regulation, and potential inhibition is critical [13].

The aim of this study is to investigate the impact of a point mutation in the amino acid sequence of the active site of M10 metallopeptidase from Streptococcus pneumoniae on its ability to coordinate with Zn(II), Ni(II), and Cu(II). The M10 metallopeptidase belongs to the MA clan and MA(M) subclan and functions as a Zn(II)-dependent MPs, as previously mentioned. Copper(II) has a higher affinity for imidazole nitrogen atoms than zinc, which suggests that it could replace Zn(II) in metallopeptidase catalytic sites. This substitution can hinder enzyme activity due to the different coordination properties of Cu(II) and Zn(II). Therefore, Cu(II) can be used as an additional strategy to enhance antibacterial efficacy against bacterial MPs. Moreover, Ni(II) also competes for the enzyme's binding site. The study investigates the binding potential of Cu(II) and Ni(II) to the target MP's metal-binding domain. Examining the coordination chemistry of Cu(II) and Ni(II) complexes with MPs may contribute to a comprehensive understanding of active and inactive metal-substituted enzymes. Recently, we have analyzed the peptide model of the original MP's metal-binding domain to determine the appropriate coordination of each metal ion with the MP's active site [24]. We discovered that the N-terminal site of the catalytic domain is preferable in metal coordination. The aim of this study is to investigate which amino acid in the N-terminus site holds greater significance in coordination, as inadequate coordination of metal ions can impede the catalytic reaction of MPs. In this paper, we examine whether substituting individual amino acids affects the metal preference of MPs. For this purpose, we determine the thermodynamic and structural properties of complexes formed by Zn(II), Ni(II), and Cu(II) ions with a mutated peptide model of the M10 metallopeptidase (Table 1). Glutamine was chosen to replace histidine and glutamic acid because of its similar biophysical properties. This amino acid is polar,hydrophilic, and structurally similar to glutamic acid. However, its sidechain cannot function as a metal binding site, which is preferable in mutation studies [25].

The research aims to determine the stability of formed complexes, identify donor atoms within the coordination sphere, and establish complex geometries at different pH. The objective is to examine the impact of point mutations on the coordination properties of the ligands, compare them with the original peptide, and identify the essential binding amino acids in the catalytic domain of the M10 protease. The findings contribute to the general understanding of the coordination chemistry of bacterial MPs with transition metal ions.