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Figure 1
MSOX movie determined at 190 K. The 75-frame structural movie captures multiple states of the enzyme catalytic cycle: (a, b and c) conversion of nitrite to nitric oxide, (d) the fully nitric oxide-bound state, (e) reformation of the water-bound resting state, and (f and g) final reduction to the copper(I)–His3 state. (a) Nitrite is bound in the `top-hat' orientation at ds1190 with a double conformation of Asp98 in the proximal and gatekeeper positions. (b) The `top-hat' nitrite immediately switches to the `side-on' orientation in ds2190 (Asp98 is present only in the proximal orientation) and nitrite only is observed until ds9190. (c) Nitric oxide is observed at ds10190 together with nitrite, (d) nitric oxide is fully present by ds18190 and is replaced by water at ds50190 and (e) water remains bound until ds69190. (f) and (g) Data sets 69–75 have no ligand bound to the T2Cu site. The 2Fobs − Fcalc density in e Å−3 is contoured at 0.57 (ds1190), 0.55 (ds2190), 0.46 (ds10190), 0.42 (ds18190), 0.40 (ds50190), 0.43 (ds69190) and 0.42 (ds75190). The cumulative X-ray dose absorbed by the crystal is indicated for each data set of the MSOX series. (h) Single-crystal UV–Vis measurements before any X-ray exposure and between X-ray data collections until ds15190. The first spectrum (red) confirmed the oxidized state of the protein crystal prior to ds1190, with absorption maxima at 456 and 578 nm. Significant reduction of the T1Cu site was observed to occur after completion of the ds1190 data collection (green spectrum), with the site predominantly reduced by ds2190. Full reduction was observed well before ds15190 (blue spectrum). Note that a progressive background shift and higher absorbance at low wavelengths is observed with continued irradiation, consistent with previous studies. (i) Dose-dependence of T1Cu reduction, using the change in absorbance at 578 nm to show the fractional change in population.

IUCrJ
Volume 5| Part 3| May 2018| Pages 283-292
ISSN: 2052-2525
https://doi.org/10.1107/S205225251800386X