Characterization of a nitrite-reducing octaheme hydroxylamine oxidoreductase that lacks the tyrosine cross-link

Abstract

The hydroxylamine oxidoreductase (HAO) family consists of octaheme proteins that harbor seven bis-His ligated electron-transferring hemes and one 5-coordinate catalytic heme with His axial ligation. Oxidative HAOs have a homotrimeric configuration with the monomers covalently attached to each other via a unique double cross-link between a Tyr residue and the catalytic heme moiety of an adjacent subunit. This cross-linked active site heme, termed the P460 cofactor, has been hypothesized to modulate enzyme reactivity toward oxidative catalysis. Conversely, the absence of this cross-link is predicted to favor reductive catalysis. However, this prediction has not been directly tested. In this study, an HAO homolog that lacks the heme-Tyr cross-link (HAOr) was purified to homogeneity from the nitrite-dependent anaerobic ammonium-oxidizing (anammox) bacterium Kuenenia stuttgartiensis, and its catalytic and spectroscopic properties were assessed. We show that HAOr reduced nitrite to nitric oxide and also reduced nitric oxide and hydroxylamine as nonphysiological substrates. In contrast, HAOr was not able to oxidize hydroxylamine or hydrazine supporting the notion that cross-link-deficient HAO enzymes are reductases. Compared with oxidative HAOs, we found that HAOr harbors an active site heme with a higher (at least 80 mV) midpoint potential and a much lower degree of porphyrin ruffling. Based on the physiology of anammox bacteria and our results, we propose that HAOr reduces nitrite to nitric oxide in vivo, providing anammox bacteria with NO, which they use to activate ammonium in the absence of oxygen.

Keywords: HAO; anammox; cytochrome c; heme; hydroxylamine oxidoreductase; nitric oxide; nitrite reductase; nitrite reduction; redox; tyrosine cross-link.

Figure 1

Electronic absorption spectra of 0.8 μM Kustc0458/7. As isolated (fully oxidized; solid black line), ascorbate reduced (dotted black line), and dithionite reduced (dashed black line). Titanium citrate addition to the isolated protein resulted in the same fully reduced spectrum (solid blue line) as seen with dithionite addition. The spectrum of as-isolated Kustc0458/7 for the 250 to 380 nm range is extracted from a different data set and has been corrected for heme concentration (at 409 nm). The inset shows a fourfold magnification of the 450 to 700 nm region

Figure 2

Reduction of nitrite to NO by HAOr. NO is produced after addition of 50 μM nitrite (at 10 min) in the presence of 200 μM phenazine ethosulfate and 100 μM ascorbate. The assay was carried out at pH 7.5 and 30 °C, and NO was measured with membrane-inlet mass spectrometry. Each colored line corresponds to a biological replicate

Figure 3

Titration of Kustc0458/7 with different substrates.A, nitrite titration of fully reduced Kustc0458/7 (1.5 μM active sites). Black to gray: 1 μM, 2 μM, 3 μM, 13 μM, 113 μM, 213 μM, 1.2 mM, and 2.2 mM. The inset shows a fourfold magnification of the 500 to 600 nm region. The Soret signal decrease is due to dilution. The peak at 314 nm corresponds to dithionite. Excess of reductant was necessary to keep Kustc0458/7 in the fully reduced state, which would get partially oxidized otherwise, even under strictly anaerobic conditions. B, electronic absorption spectra of 0.6 μM Kustc0458/7, (fully oxidized; solid black line), after addition of 2 mM hydrazine to the fully oxidized enzyme (green), after addition of 2 mM hydroxylamine to the fully oxidized enzyme (blue), after addition of 10 mM hydroxylamine to the fully reduced enzyme (pink), and fully reduced enzyme (dashed black line).

Figure 4

Potentiometric redox titration of 50 μM Kustc0458/7 performed in both reductive and oxidative directions. Global spectra Nernst fitting discerned five redox transitions; the midpoint potentials of each (with one standard deviation) and the color code used for the transitions are shown in panel C. A, ΔAbsorbance values corresponding to the Soret (420–404 nm) and the α band (552–544 nm) are independently plotted against the applied potentials and fitted to a Nernst equation with five components and fixed potentials—as discerned by the global spectra fits. Grayscale indicates different data series in either oxidizing or reductive direction during the titration, illustrating the stability of the electrochemical system throughout the experiment. Nernst fittings are shown in black lines and the points corresponding to the discerned Em values are shown as crosses. B, difference spectra corresponding to each of the five redox transitions. C, the mathematical areas corresponding to the Soret and the α bands (411–434 nm and 540–570 nm, respectively) were calculated for all discerned transitions and for the total. Assuming contribution of ten hemes to the total Soret area change and nine to the alpha band, the number of hemes contributing to each redox transition was calculated, based on both the Soret and the α band changes (color-coded bars and numbers therein). The difference between the heme count from the Soret and the α band (Δ% bar) corresponds to a fraction of one unaccounted heme, i.e., the catalytic heme.

Figure 5

Out-of-plane (OOP) heme displacements. (Sad: saddling; dom: doming; ruf: ruffling) were calculated for NeHAO (blue), KsHAO (pink), KsHDH (green), and Kustc0458/7 (orange). The inset shows a schematic representation of the heme porphyrin with each OOP distortion, where the dark circles represent atomic positions above and the light circles below the heme plane. The inset is adapted with permission from Jentzen et al. (43). Copyright 1997 American Chemical Society. The complete output can be found in the Table S1.