Abstract

Recently, Guenter Schwarz and colleagues published an elegant study in the Biochemical Journal (2019) 476, 1805–1815 which combines kinetic and spectroscopic studies with protein engineering to provide a mechanism for sulfite oxidase (SO)-catalyzed nitrite reduction that yields nitric oxide (NO). This work is noteworthy as it demonstrates that (i) for NO generation, both sulfite and nitrite must bind to the same molybdenum (Mo) center; (ii) upon sulfite reduction, Mo is reduced from +6 (MoVI) to +4 (MoIV) and MoIV reduces nitrite to NO yielding MoV; (iii) the heme moiety, linked to the Mo-center by an 11 amino acid residue tether, gets reduced by intramolecular electron transfer (IET) resulting in MoV being oxidized to MoVI; (iv) the reduced heme transfers its electron to a second nitrite molecule converting it to NO; (v) the authors demonstrate steady-state NO production in the presence of the natural electron acceptor cytochrome c; (vi) Finally, the authors use protein engineering to shorten the heme tether to reduce the heme-Mo-center distance with the aim of increasing NO production. Consequently, the rate of IET to cytochrome c is decreased but the enzymatic turnover rate for NO production is increased by ∼10-fold. This paper is unique as it provides strong evidence for a novel mechanism for steady-state NO production for human mitochondrial SO and serves as a potential template for studying NO production mechanisms in other enzymes by integrating the information gained from enzyme kinetics with EPR and UV/vis spectroscopy and protein engineering.

Nitric oxide (NO) is enzymatically generated from L-Arginine, in an oxygen-dependent manner by nitric oxide synthases (NOS) [13]. Under anaerobic conditions, NO can be generated from nitrite by NOS as well as many other metalozymes including xanthine oxidoreductase, aldehyde oxidase, nitrate reductase the mitochondrial amidoxime reductase complex and sulfite oxidase (SO) [1,48]. The subject of this commentary is a recent study [9] that aims to elucidate the catalytic mechanism of NO production by human mitochondrial SO. In this study, the authors first demonstrated the NO-release stoichiometry with the aid of an NO-analyzer. With the wild-type SO, under saturating nitrite and constant sulfite, a single turnover of the enzyme produced 2 mol of NO per monomer. Upon deletion of the heme-domain (in a variant termed SO-Mo), the NO stoichiometry dropped to 1 : 1. These results suggested that one electron upon sulfite oxidation goes the nitrite to convert it to the first molecule of NO. This results in the production of MoV, which is inert to transfer of its electron to a second molecule of nitrite. The second nitrite molecule receives the MoV-electron from the heme. As a result of intramolecular electron transfer (IET) process, MoV is oxidized back to MoVI allowing the binding of another sulfite molecule to the molybdenum center, thus starting the catalytic cycle over again. This hypothesis was proven with UV/vis spectroscopy-identifying FeII-heme and EPR spectroscopy which identified the presence of MoV.

The enzyme was also pre-reduced to MoIV with excess sulfite. After removal of sulfate and unreacted sulfite, the amount of NO generated was an order of magnitude less. This was ascribed to an H2O coordinating equatorially to the molybdenum center thus preventing efficient nitrite reduction.

Next, the authors were able to demonstrate steady-state NO generation when the heme-bound second electron was depleted by the natural electron acceptor, cytochrome c. The NO generation was also characterized as a function of [sulfite]. These experiments clearly demonstrated that sulfite was required for NO generation and that above 37 µM sulfite inhibition of NO production was observed. The kinetic analysis of this phenomenon indicated that sulfite was a competitive inhibitor, strongly indicating that, as predicted, that both sulfite and nitrite bind to the same Mo-domain.

Finally, a series of mutants of SO were generated by systematically truncating the residues spanning the heme-Moco domain, resulting in Δ5, Δ6, Δ8, Δ11-SO variants. The mutations did not affect Moco redox activity as tested by the electron acceptor, (FeIII(CN)6). The authors then used stopped-flow kinetics to determine the rate of cytochrome c reduction via IET, in the variants and the wild-type SO. The IET rate was shortened by ∼12.5-fold but the catalytic efficiency (kred/KM) increased by ∼10 fold. This meant that the second electron rather than being transferred to cytochrome c was being used to reduce the second molecule nitrite to NO.

This is an excellent mechanistic study showing the elegant manner in which SO can oxidize sulfite, and direct the electrons to reducing nitrite, to yield NO in the mitochondria. The physiological significance of SO-derived NO remains unknown, but its spatial location suggests that it may be a key regulator of mitochondrial function by NO-mediated shutdown of reactive oxygen species production under conditions of ischemia–reperfusion. In addition, this study might spur the design of therapeutic agents that enhance SO-generated NO production, in mitochondria, by blocking electron transfer from the SO-heme to cytochrome c.

Abbreviations

     
  • IET

    intramolecular electron transfer

  •  
  • NO

    nitric oxide

  •  
  • NOS

    nitric oxide synthases

  •  
  • SO

    sulfite oxidase

Competing Interests

The Author declares that there are no competing interests associated with this manuscript.

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