The high-resolution crystal structure of lobster hemocyanin shows its enzymatic capability as a phenoloxidase

Highlights

• Hemocyanin and phenoloxidase are two major members of the type 3 copper proteins.

• We determined the high-resolution crystal structure of hemocyanin from a crustacean.

• The structure of its active site is almost identical to that of phenoloxidases.

• However, hemocyanin has an active site blocker residue that is absent in phenoloxidase.

• The blocker residue would cause the functional difference between these type 3 copper proteins.

Abstract

Hemocyanin (Hc) and phenoloxidase (PO) are members of the type 3 copper protein family. Although arthropod Hc and PO exhibit similar three-dimensional structures of the copper-containing active site, Hc functions as an oxygen transport protein, showing minimal or no phenoloxidase activity. Here, we present the crystal structure of the oxy form of Hc from Panulirus japonicus (PjHc) at 1.58 Å resolution. The structure of the di-copper active site of PjHc was found to be almost identical to that of PO. Although conserved amino acids and the water molecule crucial for the enzymatic activity were observed in PjHc at almost the same positions as those in PO, PjHc showed no enzymatic activity under our experimental conditions. One striking difference between PjHc and arthropod PO was the presence of a “blocker residue” near the binuclear copper site of PjHc. This blocker residue comprised a phenylalanine residue tightly stacked with an imidazole ring of a CuA coordinated histidine and hindered substrates from accessing the active site. Our results suggest that the blocker residue is also a determining factor of the catalytic activity of type 3 copper proteins.

Introduction

Hemocyanin (Hc) is a member of the family of type 3 copper proteins, which is characterized by binuclear copper atoms, each of which is coordinated by three histidine side chains [1]. In general, Hc is abundant in the hemolymph of arthropods and mollusks, and functions as a dioxygen-transporting protein. Although Hc from both arthropods and mollusks share common structural features, namely the copper center where dioxygen is reversibly bound as peroxide in side-on bridging coordination, the structural basis of the Hc molecule as a whole between arthropods and mollusks is far less conserved [2]. Molluscan Hc forms a huge cylindrical supermolecule composed of decameric or multidecameric 330 to 450-kDa subunits. Each subunit has a subset of approximately 50-kDa sequentially arranged paralogous functional units that contain a type 3 copper center [3]. In contrast, a subunit of arthropod Hc is approximately 75-kDa and is composed of three domains, among which the binuclear copper center is found in domain II. Each subunit assembles to form a hexamer, the basic unit of arthropod hemocyanin. These hexamers sometimes gather to form a larger structure, such as a dodecamer or higher aggregates [4].

The structure of the arthropod Hc subunit is rather similar to that of arthropod phenoloxidase (PO), another member of the family of type 3 copper proteins. Arthropod proPO catalyzes the key reactions of melanin formation, i.e. the hydroxylation of monophenols to o-diphenols (monophenolase activity; E.C. 1.14.18.1) and the subsequent oxidation to the corresponding quinone (diphenolase [catecholoxidase] activity; E.C. 1.10.3.1). Proteins with those enzymatic activities are ubiquitously distributed in bacteria, fungi, plants, and vertebrates, for example, tyrosinases and polyphenoloxidases (PPO) with both mono- and di-phenolase activities, and catechol oxidases (CO) with only diphenolase activity [5,6]. The overall structures of tyrosinases, PPO, and CO from bacteria, fungi, and plants are closely related to that of the functional unit of molluscan Hc, whereas these proteins are structurally distinct from Hc and PO from arthropods [6,7]. However, the structures of the copper-containing active site are comparable among these type 3 copper proteins.

The arthropod proPO system is a major component of the innate immunity of arthropods, as quinone species (the reactive intermediate of this enzymatic reaction) are harmful to pathogenic bacteria and fungi. PO also contributes to the physical encapsulation of pathogens and wound healing by producing melanin [8]. Molecular evolutionary studies and phylogenic analyses of the primary structures of arthropod Hc and PO suggest that they are derived from a common ancestral protein with enzymatic activity, and that Hc eventually lost its PO activity and became specialized as an oxygen-transporting protein [[9], [10], [11]]. However, significant or trace PO activity of some arthropod hemocyanins has been reported [[12], [13], [14], [15], [16]].

Due to the extreme abundance and biological significance of Hc, the structural analysis of arthropod Hc has a very long history. Crustacean Hc is one of the first multimeric proteins, the crystal structure of which was solved by X-ray crystallography [[17], [18], [19], [20]]. The first determined crystal structure of Hc is from a crustacean, the California spiny lobster (Panulirus interruptus), at 3.2 Å resolution (PDB ID: 1HC1 and 1HCY) [19]. The crystal structure of arthropodan Hc from the horseshoe crab (Limulus polyphemus), an ancient arthropod that belongs to the subphylum of chelicerates, was later solved at 2.18–2.4 Å resolution (PDB ID: 1LLA, 1NOL and 1OXY) [21,22].

Although these crystal structures contributed significantly to the establishment of the mechanisms underlying the oxygen binding and transition between the oxygenated and deoxygenated states, the high-resolution crystal structure of arthropodan Hc had not been obtained to date, partially due to the complexity and heterogeneity of the Hc subunits. In contrast, the structural information of the other type 3 copper protein, POs (e.g. tyrosinases from bacteria [[23], [24], [25], [26]], PPO from plants [[27], [28], [29], [30], [31]], proPO from arthropod [[32], [33], [34]], and catecholoxidases from plants and fungi [28,35]), was obtained in recent decades. These studies have shed light on the reaction mechanisms of mono- and di-PO reactions and provided an insight into the fundamental question concerning the functional diversity among these proteins despite the striking structural similarity of their binuclear copper centers.

The importance of the conserved asparagine and glutamate residues and water molecule around the copper site has been highlighted by several research groups, based on the structural analyses of bacterial tyrosinases [5,29,36,37]. These three elements are commonly observed in tyrosinases and POs with enzymatic activity, and are suggested to be crucial elements for the o-hydroxylation of mono-phenolic substrates. Although some exceptions have been reported in plant PPOs [38,39], the significance of these elements are further supported by the structural study of arthropod PO from a mosquito [34]. In the present study, we determined the high-resolution crystal structure of oxy-form Hc from the Japanese spiny lobster (Panulirus japonicus) (PjHc). PjHc had the conserved asparagine and glutamate residues and water molecule around the copper site at almost exactly the same positions as those in the proPO structures. However, there was a striking structural difference between PjHc and a proPO from a crustacean: the blocker residue phenylalanine (Phe371) stacked with a CuA-coordinated histidine (His198) side chain. This blocker phenylalanine seems to restrict the space of the cavity around the active site and hinder the access of substrates to the copper center. This crystal structure shows the significance of the blocker residue in the enzymatic activity of type 3 copper proteins, in addition to the elements highlighted earlier.