Citation: Maiti, B.K.; Moura, J.J.G. Native Protein Template Assisted Synthesis of Non-Native Metal-Sulfur Clusters. BioChem 2022, 2, 182–197. https://doi.org/10.3390/ biochem2030013 Academic Editors: Manuel Aureliano, M. Leonor Cancela, Célia M. Antunes and Ana Cristina Rodrigues Costa Received: 21 March 2022 Accepted: 30 June 2022 Published: 1 August 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Review Native Protein Template Assisted Synthesis of Non-Native Metal-Sulfur Clusters Biplab K. Maiti 1, * and José J. G. Moura 2 1 Department of Chemistry, School of Sciences, Cluster University of Jammu, Canal Road, Jammu 180001, India 2 LAQV, REQUIMTE, Department of Chemistry, NOVA School of Sciences and Technology (FCT NOVA), 2829-516 Caparica, Portugal; jose.moura@fct.unl.pt * Correspondence: biplabmaiti@clujammu.ac.in Abstract: Metalloenzymes are the most proficient nature catalysts that are responsible for diverse biochemical transformations introducing excellent selectivity and performing at high rates, using intricate mutual relationships between metal ions and proteins. Inspired by nature, chemists started using naturally occurring proteins as templates to harbor non-native metal catalysts for the sustain- able synthesis of molecules for pharmaceutical, biotechnological and industrial purposes. Therefore, metalloenzymes are the relevant targets for the design of artificial biocatalysts. The search and devel- opment of new scaffolds capable of hosting metals with high levels of selectivity could significantly expand the scope of bio-catalysis. To meet this challenge, herein, three native scaffolds: [1Fe-4Cys] (rubredoxin), [3Fe-4S] (ferredoxin), and [S 2 MoS 2 CuS 2 MoS 2 ]-ORP (orange protein) protein scaffolds are case studies describing templates for the synthesis of non-native monomeric to mixed metal– sulfur clusters, which mimic native Ni containing metalloenzymes including [Ni-Fe] Hydrogenase and [Ni-Fe] CO Dehydrogenase. The non-native metal-substituted metalloproteins are not only useful for catalysis but also as spectroscopic probes. Keywords: designed metalloproteins; models of [Ni-Fe]-hydrogenase and [Ni-Fe]-CODH; orange- protein and spectroscopic probes 1. Introduction Nature has evolved in order to utilize metal ions and/or metal clusters within protein scaffolds to build up metalloproteins that accomplish diverse chemical reactions enabling to sustain of life [1–4]. The versatility of the metals and biological ligands available in proteins is amazing. The same metal (with a set of conserved amino acids as ligands) may show different electronic/physical properties, performing a wide range of biological roles in different metalloproteins [5–7]. Nature utilizes a range of different metals and recruits the correct metal into proper protein environments to execute selective functions [5–7]. The nuclearity of metal-cofactors varies from monomeric to multimeric. Monomeric metalloenzymes are well studied, such as cupredoxin [8], rubredoxin [9], cytochrome P450 [10,11], and molybdenum-enzymes [12], which are involved in a variety of biochemical transformations, and with relevant roles in electron transfer processes. Furthermore, many biochemical transformations occurred by a variety of complex metalloenzymes such as nitrogen-fixing nitrogenases [13,14], photosystem [15,16], hydrogenases [17,18], and carbon monoxide- dehydrogenase (CODH) [19,20]. However, many enzymes show intrinsic promiscuity [21,22] for various forms of chemical reactions, whereas other activities are obtained by only a small alteration of their active site or protein environment [23]. The diversity of promiscuous enzymatic activity can be expanded by the incorporation of a variety of metallic ions at the active sites of metalloproteins, catalyzing a wide range of chemical transformations [2,24–26]. Handling of the metal-binding site of metalloprotein is usually aimed for two main reasons: (i) to replicate the active site of other native metalloenzymes, and (ii) to design spectroscopic BioChem 2022, 2, 182–197. https://doi.org/10.3390/biochem2030013 https://www.mdpi.com/journal/biochem