Motion of proximal histidine and structural allosteric transition in soluble guanylate cyclase

Abstract

We investigated the changes of heme coordination in purified soluble guanylate cyclase (sGC) by time-resolved spectroscopy in a time range encompassing 11 orders of magnitude (from 1 ps to 0.2 s). After dissociation, NO either recombines geminately to the 4-coordinate (4c) heme (τ G1 = 7.5 ps; 97 ± 1% of the population) or exits the heme pocket (3 ± 1%). The proximal His rebinds to the 4c heme with a 70-ps time constant. Then, NO is distributed in two approximately equal populations (1.5%). One geminately rebinds to the 5c heme (τ G2 = 6.5 ns), whereas the other diffuses out to the solution, from where it rebinds bimolecularly (τ = 50 μs with [NO] = 200 μM) forming a 6c heme with a diffusion-limited rate constant of 2 × 10 8 M −1 ·s −1 . In both cases, the rebinding of NO induces the cleavage of the Fe-His bond that can be observed as an individual reaction step. Saliently, the time constant of bond cleavage differs depending on whether NO binds geminately or from solution (τ 5C1 = 0.66 μs and τ 5C2 = 10 ms, respectively). Because the same event occurs with rates separated by four orders of magnitude, this measurement implies that sGC is in different structural states in both cases, having different strain exerted on the Fe-His bond. We show here that this structural allosteric transition takes place in the range 1-50 μs. In this context, the detection of NO binding to the proximal side of sGC heme is discussed.soluble guanylate cyclase | nitric oxide | time-resolved absorption spectroscopy | allostery | protein activation T he soluble guanylate cyclase (sGC), localized in many different cell types, is the receptor of the endogenous messenger nitric oxide (NO) and catalyzes the formation of cGMP from GTP upon activation triggered by NO binding (1, 2). The diatomic messenger NO and sGC play a critical role in several physiological processes: regulation of vascular blood pressure and cardiovascular diseases (3), lung airway relaxation and pulmonary pathologies (4), immune response and inflammatory disorders (5), and tumor progression and apoptosis (6). Thus, sGC is a pharmacological target of very high interest, and several activators have been developed (7,8), leading to the approval of riociguat for the treatment of pulmonary hypertension (9, 10). Because of its pharmacological interest, the mechanisms of activation, deactivation, and regulation of sGC must be deciphered at the molecular level. Despite numerous efforts, the 3D crystal structure of heterodimeric sGC remains unknown, but the heme domain of the sGC β1-subunit [called heme NO/oxygen-binding (H-NOX)] was modeled from the heme domain of bacterial NO sensors (11,12) and the sGC catalytic α1-subunit was modeled from the catalytic α1-subunit of adenylate cyclase (13). Recently, the entire quaternary structure of sGC was reconstructed by inserting individual protein domains into the density envelope of entire single-sGC molecules observed by EM (14), revealing a high flexibility of the sGC dimer. Subsequently, the structural perturbations induced by NO bi...

Tissue‐specific regulation of cytochrome c by post‐translational modifications: respiration, the mitochondrial membrane potential, ROS, and apoptosis

“…The researchers concluded that nitrosylation of Cytc may play a role in apoptosis regulation (144). Endogenous heme nitrosylation is a fairly rare event that is observed in only a few other proteins, such as COX and guanylyl cyclase, the only known receptor for NO (145)(146)(147)(148). In both COX and guanylyl cyclase, the heme iron is pentacoordinated, unlike in Cytc, where it is hexacoordinated (149).…”

Structural Insight into H‐NOX Gas Sensing and Cognate Signaling Protein Regulation

“…Yoo et al. used time‐resolved absorption spectroscopy to study the NO binding kinetics of full‐length sGC and provided support for the distal‐NO complex model on short timescales (recently reviewed). The spectral observations could be described without invoking a dinitrosyl intermediate or proximal‐NO complex.…”

Structural changes and picosecond to second dynamics of cytochrome c in interaction with nitric oxide in ferrous and ferric redox states

“…Although the geminate yield is as large as that measured for 4c-hemes (98.5%) it differentiates from these proteins by the presence of two geminate components, as observed for myoglobin 72 and for the L16A mutant of the bacterial NO-carrier cytochrome c' from Alcaligenes xylosoxidans. 73 In both redox states NO geminate rebinding occurs with a second phase having same time constant (55 ps) and a small proportion of NO exiting the heme pocket (1.5 -3.5%).…”