UC Berkeley

The Heme-Nitric oxide/OXygen binding (H-NOX) domain encompasses a family of proteins closely related (≤ 40% sequence identity) to the heme domain of soluble guanylate cyclase (sGC), the eukaryotic enzyme receptor for NO [1, 2]. sGC discriminates between NO and O₂, and exclusively binds NO even in the presence of excess O₂. Although some H-NOX domains have ligand binding properties that are identical to sGC, others additionally bind O₂, including the atypical sGCs from Drosophila melanogaster and Caenorhabditis elegans [1, 3, 4]. The molecular basis of this ligand selectivity is not fully understood. In order to better understand the H-NOX domain, resonance Raman (RR) spectroscopy was employed in combination with isotopic substitution and site-directed mutagenesis to probe the heme pocket in Thermoanaerobacter tengcongensis (Tt H-NOX) as a model system for this family of heme proteins. Thus, the expression, purification, and RR characterization of Tt H-NOX and selected mutants were performed to elucidate the heme environmental properties that influence ligand binding.

A striking feature of the O₂-bound WT Tt H-NOX crystal structure is the presence of a distorted heme molecule [5]. RR investigation of Tt H-NOX proteins containing mutations at key conserved residues, Ile-5 and Pro-115, determined that the most dramatic heme conformational changes occurred in the O₂-bound forms, and that the single P115A mutation generated a significantly relaxed chromophore. Decreased RR intensities were observed for several out-of-plane modes in the 400-750 cm^-1 region known to be sensitive to ruffling and saddling deformations, as well as increased vibrational frequencies for the core heme skeletal modes. These changes demonstrated that the P115A heme conformation was considerably more relaxed than WT, with increased flexibility within the protein pocket that allowed for rapid sampling of alternate conformations.

Another remarkable feature of this family is the spectroscopic observation of an unusually high ν(C-O) frequency. To elucidate the interactions responsible for this property, mutations were made in Tt H-NOX to probe the distal pocket, the conserved Tyr-Ser-Arg (YxSxR) motif, and the heme-linkage site (His-102). RR spectra of these mutants indicated that H-bonding interactions between these residues and the heme significantly affected the CO bond by increasing the back-donation of the Fe^II d_π electrons into the CO π^* orbitals. The most significant change occurred upon disruption of the H-bonds between the YxSxR motif and the heme propionate groups, producing two dominant CO-bound heme conformations; one was structurally similar to WT, and the other conformer displayed ν(C-O) downshifts of up to ~70 cm^-1. Based on these shifts, the most important factor contributing to the C-O stretching mode may be the neutralization of the negative charges on the propionate groups via the strictly conserved YxSxR motif.

Finally, the role of the Tt H-NOX binding pocket in stabilizing the O₂ complex was investigated. Evidence of H-bonds to the bound O₂ was demonstrated in the RR spectra of Trp-9 and Tyr-140 mutants; the disruption of the H-bond network in these mutations increased ν(Fe-O₂) upon returning electron density to the Fe-O₂ bond. In contrast, the addition of steric bulk to the H-NOX pocket decreased the ν(Fe-O₂) frequency relative to the WT protein. These shifts suggested that the bulky distal residues forced the H-NOX binding pocket into a more open position that increased the distance between Tyr-140 and the O₂, thereby weakening this crucial H-bond. Thus, two distinct factors influence the ν(Fe-O₂) in Tt H-NOX: (i) electrostatic effects from H-bonding with Tyr-140, which increased the ν(Fe-O₂) frequency upon its removal; and (ii) steric effects which decreased ν(Fe-O₂) due to bond lengthening from a more loosely held O₂ ligand.

In summary, this work presents a comparative analysis of the heme environmental effects in Tt H-NOX. By directly probing the interactions between the heme chromophore and the protein pocket through a combination of RR spectroscopy and site-directed mutagenesis, this study may elucidate the biochemical properties of these H-NOX domains, and bring insight into the relationship between heme structure and protein function in this family.