A Review of the Catalytic Mechanism of Human Manganese Superoxide Dismutase

Abstract

Superoxide dismutases (SODs) are necessary antioxidant enzymes that protect cells from reactive oxygen species (ROS). Decreased levels of SODs or mutations that affect their catalytic activity have serious phenotypic consequences. SODs perform their bio-protective role by converting superoxide into oxygen and hydrogen peroxide by cyclic oxidation and reduction reactions with the active site metal. Mutations of SODs can cause cancer of the lung, colon, and lymphatic system, as well as neurodegenerative diseases such as Parkinson's disease and amyotrophic lateral sclerosis. While SODs have proven to be of significant biological importance since their discovery in 1968, the mechanistic nature of their catalytic function remains elusive. Extensive investigations with a multitude of approaches have tried to unveil the catalytic workings of SODs, but experimental limitations have impeded direct observations of the mechanism. Here, we focus on human MnSOD, the most significant enzyme in protecting against ROS in the human body. Human MnSOD resides in the mitochondrial matrix, the location of up to 90% of cellular ROS generation. We review the current knowledge of the MnSOD enzymatic mechanism and ongoing studies into solving the remaining mysteries.

Keywords: anti-oxidant; catalysis; human; manganese; mechanism; mitochondria; reactive oxygen species; superoxide dismutase.

Figure 1

Human MnSOD. (a) Each subunit contains a manganese ion at the catalytic center, indicated by pink spheres. (b) The active site. Red spheres denote oxygen atoms, blue denotes nitrogen atoms, grey denotes carbon atoms from one subunit of the tetramer, and magenta denotes carbon atoms from the adjacent subunit. The dashed lines represent the hydrogen bond network hypothesized to be the proton relay to the manganese ion used for catalysis. Glu162 hydrogen bonds with His163 across the dimer interface. Adapted from Azadmanesh et al., 2017, PDB entry 5VF9 [44]. WAT1: single oxygen-containing molecule; WAT2: single oxygen-containing molecule. Single letter amino acid code is used.

Figure 2

Electrostatic surfaces of human MnSOD. (a) The electrostatic surface of the tetramer with only one active site viewed in this orientation (yellow arrow and dashed lines). (b) A zoomed-in view of the dimer interface indicated by a yellow, dashed line. The labels for Glu162 and Arg173 are white to indicate their location on the concave surface within the active site pit. (c) A cross section-view of the active site across the dimer interface, rotated 90° along the horizontal axis in relation to (a,b). Glu162 is behind Arg173 in this view. Electrostatic surface is colored in kiloteslas. Adapted from Azadmanesh et al., 2017 [44]. Single letter amino acid code is used.

Figure 3

Human MnSOD 5-6-5 mechanism adapted from Lah et al., 1995 [59]. Dotted lines indicated hydrogen bonds.

Figure 4

Protonation state of His30 in human MnSOD revealed by neutron diffraction. Carbons are green, nitrogens are blue, and oxygens are red. Deuterium (yellow) replaced hydrogen in the sample to increase diffraction signal. The 2F_o-F_c grey nuclear density is 1.0 σ. The red omit positive F_o-F_C nuclear density is at 2.5 σ. (a) Chain A of the neutron crystal structure displays an unprotonated His30. (b) Chain B shows a protonated His30. Methods and statistics for crystal growth, deuterium exchange, data collection, and data reduction are found in Azadmanesh et al., 2017 [84]. Data was refined to 2.30 Å using PHENIX.REFINE with R_work and R_free values of 0.28, and 0.31, respectively (unpublished) [85,86].

Figure 5

Putative superoxide-dependent proton shuttling mechanism. Black dashed lines indicate the proton transfer relay during catalysis. Red dashed line indicates the proton transfer from WAT1 to Asp159 that results in the inactive complex whereas the backwards transfer restores activity of the enzyme (model based on PDB entry 5T30). Green dashed lines indicate stabilizing hydrogen-bond interactions for molecules of the proton relay. Of note, superoxide is thought to bind Mn³⁺ directly but not Mn²⁺, where it is instead bound to the active site solely through hydrogen bonding to members of the hydrogen-bond network. Single letter amino acid code is used.