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Published in final edited form as: Biochemistry. 2012 Mar 19;51(13):2737–2746. doi: 10.1021/bi300105s

(A).Time-resolved spectra of the reaction between sGC and stoichiometric amount of NO. NO binding reactions of sGC monitored by rapid-scan single mixing at 24 °C. Both sGC and NO were at 7 µM. Three spectra recorded at 1.3 ms, 20 ms and 1.0 s out of the 400 collected are shown. A matching resting Fe(II) sGC spectrum collected on a HP8453 spectrophotometer is also shown as "zero" time control (dashed line). (B). Transient formation of 6c NO-sGC identified by RFQ EPR kinetic measurement. 10 µM each of sGC and NO was reacted at 24 °C by single-stage equal mixing and freeze trapped by cold isopentane (113 K). The ram velocity was 1.25 cm/s and the packing factor was 0.45 (16). The EPR spectra for samples trapped at 20 ms, (b), and 5 s, (a) are one out of three repeats, which show similar results. Spectrum (c) is the difference between (b) and 25% of (a) to reveal the dominant 6c NO-sGC species. Simulation of the EPR of 6c NO-sGC, spectrum (d), was done by Simfonia as frozen powder sample. Hyperfine splitting constants related to the nitrogen nuclei from NO, A_N1, and His105, A_N2, are indicated. Spectra (a) and (b) are the average of 32 scans. Key g values for the NO-sGC complexes are also indicated. (C). Kinetics of the resting sGC (by A_{432 nm}), 6c NO-sGC (A_{420 nm}) and 5c NO-sGC (A_{399 nm}) in the second stage reaction of the anaerobic sequential stopped flow at 24 °C. Reaction in the first stage was between 5 µM sGC and 5 µM NO, and the mixture, after 20 ms delay, was further reacted with 5 µM NO in the 2nd mixing. Dashed lines are visual guide connecting all data points which are not evenly distributed in logarithmic scale, especially those < 10 ms.

(A).Time-resolved spectra of the reaction between sGC and stoichiometric amount of NO. NO binding reactions of sGC monitored by rapid-scan single mixing at 24 °C. Both sGC and NO were at 7 µM. Three spectra recorded at 1.3 ms, 20 ms and 1.0 s out of the 400 collected are shown. A matching resting Fe(II) sGC spectrum collected on a HP8453 spectrophotometer is also shown as "zero" time control (dashed line). (B). Transient formation of 6c NO-sGC identified by RFQ EPR kinetic measurement. 10 µM each of sGC and NO was reacted at 24 °C by single-stage equal mixing and freeze trapped by cold isopentane (113 K). The ram velocity was 1.25 cm/s and the packing factor was 0.45 (16). The EPR spectra for samples trapped at 20 ms, (b), and 5 s, (a) are one out of three repeats, which show similar results. Spectrum (c) is the difference between (b) and 25% of (a) to reveal the dominant 6c NO-sGC species. Simulation of the EPR of 6c NO-sGC, spectrum (d), was done by Simfonia as frozen powder sample. Hyperfine splitting constants related to the nitrogen nuclei from NO, A_N1, and His105, A_N2, are indicated. Spectra (a) and (b) are the average of 32 scans. Key g values for the NO-sGC complexes are also indicated. (C). Kinetics of the resting sGC (by A_{432 nm}), 6c NO-sGC (A_{420 nm}) and 5c NO-sGC (A_{399 nm}) in the second stage reaction of the anaerobic sequential stopped flow at 24 °C. Reaction in the first stage was between 5 µM sGC and 5 µM NO, and the mixture, after 20 ms delay, was further reacted with 5 µM NO in the 2nd mixing. Dashed lines are visual guide connecting all data points which are not evenly distributed in logarithmic scale, especially those < 10 ms.